Publications by authors named "Esther García-Rosado"

22 Publications

  • Page 1 of 1

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

Gene 2021 Mar 11;774:145430. Epub 2021 Jan 11.

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

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gene.2021.145430DOI Listing
March 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cimid.2020.101426DOI Listing
April 2020

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo.

J Fish Dis 2019 Feb 3;42(2):221-227. Epub 2018 Dec 3.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV-type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C-terminal side of the capsid. However, the concurrent mutation in the N-terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C-terminal and N-terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double- and triple-mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C-terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N-terminal side is also important for viral replication in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.12916DOI Listing
February 2019

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses.

Fish Shellfish Immunol 2018 Dec 7;83:148-157. Epub 2018 Sep 7.

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

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2018.09.022DOI Listing
December 2018

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Front Immunol 2018 17;9:1626. Epub 2018 Jul 17.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Malaga, Malaga, Spain.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression () and IFN-stimulated genes (, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.01626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056728PMC
July 2018

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

Fish Shellfish Immunol 2018 Jul 25;78:270-278. Epub 2018 Apr 25.

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

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2018.04.054DOI Listing
July 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2017.05.040DOI Listing
July 2017

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish Shellfish Immunol 2016 Aug 29;55:642-6. Epub 2016 Jun 29.

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

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2016.06.043DOI Listing
August 2016

SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system.

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

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

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13567-015-0304-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705746PMC
January 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2013.09.016DOI Listing
November 2013

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass(Dicentrarchus labrax) during the course of an experimental challenge.

J Vet Sci 2012 Dec;13(4):355-62

IFAPA Centro El Toruño, Junta de Andalucía. El Puerto de Santa María, Cádiz, Spain.

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539120PMC
http://dx.doi.org/10.4142/jvs.2012.13.4.355DOI Listing
December 2012

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10126-012-9478-8DOI Listing
April 2013

Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax).

Vet Microbiol 2011 Dec 1;154(1-2):86-95. Epub 2011 Jul 1.

IFAPA Centro El Toruño, Junta de Andalucía. Ctra N.IV, Camino de Tiro Pichón, C.P.: 11.500, El Puerto de Santa María, Cádiz, Spain.

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2011.06.029DOI Listing
December 2011

Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity.

Virus Res 2008 May 4;133(2):228-38. Epub 2008 Mar 4.

Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway.

In this study we characterize two proteins encoded by the two smallest genomic segments of the piscine orthomyxovirus infectious salmon anaemia virus (ISAV). Both proteins, encoded by the un-spliced ORF from genomic segment 7 (s7ORF1) and the larger ORF from segment 8 (s8ORF2), are involved in modulation of the type I interferon (IFN) response. The data suggests that the s7ORF1 protein is collinearly encoded, non-structural, contains no nuclear localisation signals, localises mainly to the cytoplasmic perinuclear area and does not bind single- or double-stranded RNA. On the other hand, genomic segment 8 uses a bicistronic coding strategy and the encoded s8ORF2 protein is a structural component of the viral particle. This protein contains two nuclear localisation signals, has a predominantly nuclear localisation, binds both double-stranded RNA and poly-A tailed single-stranded RNA, but not double-stranded DNA. In poly I:C stimulated salmon cells both ISAV proteins independently down-regulate the type I IFN promoter activity. Thus, ISAV counteracts the type I IFN response by the action of at least two of its gene products, rather than just one, as appears to be the case for other known members of the Orthomyxoviridae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2008.01.008DOI Listing
May 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.fsi.2007.10.010DOI Listing
February 2008

Co-occurrence of viral and bacterial pathogens in disease outbreaks affecting newly cultured sparid fish.

Int Microbiol 2007 Sep;10(3):193-9

Department of Microbiology, University of Málaga, Málaga, Spain.

Several microbial disease outbreaks in farm stocks of newly cultured sparid fish species, such as common seabream, redbanded seabream, and white seabream, were recorded from 2004 to 2006. This study describes the isolation and characterization of the potential causative agents, either bacteria or viruses, of these outbreaks. The isolated bacterial strains were characterized according to traditional taxonomical analyses and sequencing of a 16S rDNA fragment. Most bacteria were identified as Vibrio spp. and Photobacterium damselae subsp. damselae. The development of cytopathic effects (CPE) on different fish cell lines, the application of specific nested-PCR tests for infectious pancreatic necrosis virus (IPNV), viral nervous necrosis virus (VNNV) and viral hemorrhagic septicemia virus (VHSV), and subsequent sequence analyses were used for virus detection and identification. VNNV, related to the striped jack neural necrosis virus (SJNNV) genotype, and VHSV, related to the genotype Ia, were the only viruses detected. VNNV was isolated from the three fish species under study in five different outbreaks, whereas VHSV was isolated from common seabream and white seabream during two of these outbreaks. IPNV was not detected in any case.
View Article and Find Full Text PDF

Download full-text PDF

Source
September 2007

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetimm.2007.09.008DOI Listing
January 2008

Protein and glycoprotein content of lymphocystis disease virus (LCDV).

Int Microbiol 2004 Jun;7(2):121-6

Department of Microbiology, Faculty of Sciences, University of Malaga, Spain.

The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
June 2004