RESUMO
IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.
Assuntos
Bass , Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Antivirais , Bass/genética , Citocinas/genética , Nodaviridae/genéticaRESUMO
Aquaculture constitutes an alternative source for food production and contributes to a reduction in the indiscriminate catching of aquatic organisms in their natural environment. However, high mortality during the larval state remains a challenge in this sector, mainly because of factors such as diet and diseases caused by pathogens. Therefore, growth and health management is a key strategy for sustainable aquaculture. Synthetic growth hormone secretagogues (GHSs) are a family of ligands that can stimulate pituitary growth hormone release as well as the function of ghrelin, contributing to the immune responses in a variety of vertebrates, including fish. The A233 decapeptide is a GHS with a demonstrated impact on growth, immune system function, and antioxidant defense in tilapia fish, but no antiviral activity has been described for this peptide. Here, using an in vitro model (TRG-2 cells) and two in vivo models (sea bream [Sparus aurata]) and zebrafish [Danio rerio]), we demonstrate for the first time the potential antiviral effect of A233 in teleost fish.
Assuntos
Grelina , Dourada , Animais , Grelina/farmacologia , Hormônio do Crescimento/metabolismo , Secretagogos , Peixe-Zebra/metabolismoRESUMO
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 (Mut247+270Dl956, 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 Mut247+270Dl956 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.
Assuntos
Bass/genética , Doenças dos Peixes/imunologia , Nodaviridae/fisiologia , Nodaviridae/patogenicidade , Infecções por Vírus de RNA/veterinária , Transcriptoma/imunologia , Animais , Bass/imunologia , Encéfalo/virologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica/veterinária , Rim Cefálico/virologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/microbiologia , VirulênciaRESUMO
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-1â¯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-1â¯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-1â¯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.
Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interferon Tipo I/genética , Dourada/genética , Dourada/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interferon Tipo I/química , Interferon Tipo I/imunologia , Nodaviridae/fisiologia , Novirhabdovirus/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Rhabdoviridae/imunologia , Alinhamento de Sequência/veterináriaRESUMO
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.
Assuntos
Citocinas/genética , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Linguados , Nodaviridae/fisiologia , Infecções por Vírus de RNA/veterinária , Ubiquitinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Citocinas/química , Citocinas/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Nodaviridae/genética , Filogenia , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMO
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.
Assuntos
Doenças dos Peixes/virologia , Linguados , Interferons/metabolismo , Novirhabdovirus/imunologia , Infecções por Rhabdoviridae/veterinária , Animais , Doenças dos Peixes/imunologia , Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral , Novirhabdovirus/genética , Poli I-C , RNA Viral , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Replicação ViralRESUMO
Gilthead sea bream and European sea bass display different resistance-susceptibility patterns during infection with different nervous necrosis virus (NNV) species, which may derive from differences in the triggered immune response. Based on this premise, we analysed the transcription of several selected immune-related genes in sea bream experimentally infected with NNV isolates obtained from sea bass (DlNNV, RGNNV) or sea bream (SaNNV, RGNNV/SJNNV). Viral replication only occurred in SaNNV-inoculated fish; therefore, the differences between the immune response elicited by both viruses may be the key to understanding the mechanism behind the inhibition of DlNNV replication. Principal component analysis clustered samples according to the viral isolate from 1 day post infection onwards and evidenced differences in the immune response against both viruses, even though no mortalities or symptoms were recorded. The response against DlNNV is characterized by higher rtp3 transcription early after the infection, longer-lasting il-10 transcription and stronger induction of casp1 and hsp70. These genes should be targets for future studies in order to elucidate their role in hampering NNV replication in sea bream, which is essential for developing effective prophylactic measures.
RESUMO
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.
Assuntos
Linguados/genética , Componentes do Gene/genética , Regulação da Expressão Gênica/genética , Proteínas de Resistência a Myxovirus/genética , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , Ilhas de CpG/genética , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Oncorhynchus mykiss , Plasmídeos/genética , Poli I-C/farmacologia , Elementos de Resposta/genética , SalmãoRESUMO
Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.
Assuntos
Bass , Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Bass/genética , Lipopolissacarídeos , Genômica , Genótipo , Poli I-C/farmacologia , Doenças dos Peixes/genética , Nodaviridae/genéticaRESUMO
Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.
RESUMO
The use of functional feeds in aquaculture is currently increasing. This study aimed to assess the combined impact of dietary green microalgae Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (CVP diet) on thick-lipped grey mullet (Chelon labrosus) juvenile fish. The effects on intestinal microbiota and the transcription of genes related to metabolism, stress, and the immune system were investigated after 90 days of feeding. Additionally, the fish were challenged with Aeromonas hydrophila and polyinosinic-polycytidylic acid (poly I:C) to evaluate the immune response. Microbiota analysis revealed no significant differences in alpha and beta diversity between the anterior and posterior intestinal sections of fish fed the control (CT) and CVP diets. The dominant genera varied between the groups; Pseudomonas and Brevinema were most abundant in the CVP group, whereas Brevinema, Cetobacterium, and Pseudomonas were predominant in the CT group. However, microbial functionality remained unaltered. Gene expression analysis indicated notable changes in hif3α, mhcII, abcb1, mx, and tnfα genes in different fish organs on the CVP diet. In the head kidney, gene expression variations were observed following challenges with A. hydrophila or poly I:C, with higher peak values seen in fish injected with poly I:C. Moreover, c3 mRNA levels were significantly up-regulated in the CVP group 72 h post-A. hydrophila challenge. To conclude, incorporating C. fusca with V. proteolyticus in C. labrosus diet affected the microbial species composition in the intestine while preserving its functionality. In terms of gene expression, the combined diet effectively regulated the transcription of stress and immune-related genes, suggesting potential enhancement of fish resistance against stress and infections.
RESUMO
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.
Assuntos
Antibiose , Água Doce/virologia , Novirhabdovirus/fisiologia , Água do Mar/virologia , Microbiologia da Água , Genes Virais , Genoma Viral , Novirhabdovirus/isolamento & purificação , Novirhabdovirus/patogenicidade , Regiões Promotoras Genéticas , Transcrição Gênica , Replicação ViralRESUMO
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.
Assuntos
Aquabirnavirus/imunologia , Infecções por Birnaviridae/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/biossíntese , Proteínas de Ligação ao GTP/biossíntese , Animais , Aquabirnavirus/genética , Aquabirnavirus/fisiologia , Aquicultura , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Doenças dos Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Linguados , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Proteínas de Resistência a Myxovirus , Poli I-C/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Dourada , Transcrição Gênica , Replicação ViralRESUMO
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.
Assuntos
Biotecnologia/métodos , Células-Tronco Embrionárias , Peixes , Técnicas de Transferência de Genes/veterinária , Animais , Animais Geneticamente Modificados , Técnicas de Cultura de Células , Quimera , Pesqueiros , Peixes/embriologia , Peixes/genética , Técnicas de Transferência de Genes/tendências , Camundongos , Modelos AnimaisRESUMO
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.
Assuntos
Linhagem Celular/citologia , DNA/metabolismo , Dourada/genética , Animais , Genótipo , Hibridização in Situ Fluorescente , Cariotipagem , Repetições de Microssatélites/genética , RNA Ribossômico/genética , Telomerase/metabolismoRESUMO
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.
Assuntos
Linguados/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Vírus da Doença Hemorrágica Epizoótica/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Proteínas de Resistência a Myxovirus/genética , Novirhabdovirus/fisiologia , Oncorhynchus mykiss , Regiões Promotoras Genéticas , Salmão/embriologia , TransfecçãoRESUMO
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.
Assuntos
Linguados/metabolismo , Proteínas de Ligação ao GTP/farmacologia , Vírus da Necrose Pancreática Infecciosa/efeitos dos fármacos , Novirhabdovirus/efeitos dos fármacos , Ranavirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Primers do DNA/genética , DNA Complementar/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Resistência a Myxovirus , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmão , TransfecçãoRESUMO
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.
Assuntos
Encéfalo/imunologia , Portador Sadio/veterinária , Doenças dos Peixes/imunologia , Infecções por Vírus de RNA/veterinária , Dourada , Animais , Linfócitos B/imunologia , Encéfalo/patologia , Encéfalo/virologia , Citocinas/imunologia , Doenças dos Peixes/virologia , Rim Cefálico/imunologia , Imunoglobulina M/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologiaRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/genética , Peixes/metabolismo , Técnicas de Transferência de Genes , Transposases/metabolismo , Animais , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Peixes/genética , Instabilidade Genômica/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Transposases/genéticaRESUMO
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.