RESUMEN

The African bichir (Polypterus senegalus) is a living representative of Polypteriformes. P. senegalus possesses teeth composed of dentin covered by an enameloid cap and a layer of collar enamel on the tooth shaft, as in lepisosteids. A thin layer of enamel matrix can also be found covering the cap enameloid after its maturation and during the collar enamel formation. Teleosts fish do not possess enamel; teeth are protected by cap and collar enameloid, and inversely in sarcopterygians, where teeth are only covered by enamel, with the exception of the cap enameloid in teeth of larval urodeles. The presence of enameloid and enamel in the teeth of the same organism is an opportunity to solve the evolutionary history of the presence of enamel/enameloid in basal actinopterygians. In silico analyses of the jaw transcriptome of a juvenile bichir provided twenty SCPP transcripts. They included enamel, dentin, and bone-specific SCPPs known in sarcopterygians and several actinopterygian-specific SCPPs. The expression of these 20 genes was investigated by in situ hybridizations on jaw sections during tooth and dentary bone formation. A spatiotemporal expression patterns were established and compared with previous studies of SCPP gene expression during enamel/enameloid and bone formation. Similarities and differences were highlighted, and several SCPP transcripts were found specifically expressed during tooth or bone formation suggesting either conserved or new functions of these SCPPs.

Asunto(s)

Calcificación Fisiológica , Diente , Animales , Calcificación Fisiológica/genética , Senegal , Diente/metabolismo , Peces/genética , Evolución Biológica

RESUMEN

The understanding of virus-host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection.

Asunto(s)

Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/virología , Virus de la Necrosis Pancreática Infecciosa , Proteínas de Resistencia a Mixovirus/genética , Novirhabdovirus , Infecciones por Rhabdoviridae/veterinaria , Dorada , Animales , Infecciones por Birnaviridae/fisiopatología , Línea Celular , Enfermedades de los Peces/genética , Regiones Promotoras Genéticas , Transfección/veterinaria

RESUMEN

Due to their direct antiviral activity, Mx proteins play a main role in the response mediated by type I interferon against viral infections. The study on gilthead seabream Mx proteins is especially interesting, since this species is unusually resistant to viral diseases, being asymptomatic carrier of several viruses pathogenic to other fish species. Gilthead seabream has three Mx proteins (Mx1, Mx2 and Mx3) that, separately, display antiviral activity against a wide range of viruses, showing interesting differences in their antiviral specificities. In this work, the possible synergy between the three Mx isoforms has been studied using in vitro systems consisting of CHSE-214 cells stably expressing two or the three gilthead seabream Mx proteins. The antiviral activity of these Mx combinations has been tested against the Infectious Pancreatic Necrosis Virus (IPNV), the Viral Haemorrhagic Septicaemia Virus (VHSV), the European Sheatfish Virus (ESV) and the Lymphocystis Disease Virus (LCDV). A synergistic effect of the Mx proteins was only detected against ESV, no synergy was observed against LCDV, and a negative interference was detected against the two RNA viruses tested, IPNV and VHSV, as viral replication was higher in cells expressing certain Mx combinations than in cells expressing these proteins separately. These results suggest a functional interaction between gilthead seabream Mx isoforms, which results in a higher or lower antiviral activity depending on the virus tested, thus supporting the idea of complex virus-host interactions and finely tuned mechanisms controlling the antiviral activity of Mx proteins.

Asunto(s)

Antivirales/inmunología , Proteínas de Peces/inmunología , Proteínas de Resistencia a Mixovirus/inmunología , Dorada/inmunología , Animales , Línea Celular , Efecto Citopatogénico Viral , Proteínas de Peces/genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Virus de la Necrosis Pancreática Infecciosa/inmunología , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , Iridoviridae/inmunología , Iridoviridae/patogenicidad , Proteínas de Resistencia a Mixovirus/genética , Novirhabdovirus/inmunología , Novirhabdovirus/patogenicidad , Ranavirus/inmunología , Ranavirus/patogenicidad , Dorada/genética , Dorada/virología

RESUMEN

Mx proteins are important components of the antiviral innate immune response mediated by type I interferon. Classically, these proteins have been considered to be triggered by viral RNA, thus showing activity against RNA viruses. Actually, three Mx proteins (SauMx1, SauMx2 and SauMx3) from gilthead seabream (Sparus aurata) have previously shown antiviral activity against a dsRNA virus: the infectious pancreatic necrosis virus (IPNV) in vitro. For further characterizing their antiviral spectrum, the activity of SauMx proteins were tested against three different viral pathogens of fish: the lymphocystis disease virus (LCDV, a dsDNA virus), a pathogen of gilthead seabream; the viral haemorrhagic septicaemia virus (VHSV, a ssRNA virus), to which gilthead seabream is considered a reservoir species; and the European sheatfish virus (ESV, a dsDNA virus), that has not been detected in gilthead seabream to date. Three clonal populations of CHSE-214 cells developed in a previous study, stably expressing SauMx1, SauMx2 and SauMx3, respectively, were challenged with the three viruses. Results combining cytopathic effects and virus yield reduction assays showed that SauMx1 protected the cells against VHSV and LCDV, SauMx2 protected against ESV and LCDV, and SauMx3 showed activity only against VHSV. This study, besides confirming the antiviral activity of the three gilthead seabream Mx proteins, is the first report of the protective effect of a fish Mx against DNA viruses. Additionally, it discloses a clear specificity between Mx proteins and virus targets, supporting the idea that the relationship between virus and Mx proteins is finely tuned.

Asunto(s)

Virus ADN/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Proteínas de Resistencia a Mixovirus/inmunología , Virus ARN/inmunología , Dorada/inmunología , Animales , Línea Celular , Virus ADN/fisiología , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Virus de la Necrosis Pancreática Infecciosa/inmunología , Virus de la Necrosis Pancreática Infecciosa/fisiología , Iridoviridae/inmunología , Iridoviridae/fisiología , Proteínas de Resistencia a Mixovirus/genética , Novirhabdovirus/inmunología , Novirhabdovirus/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Virus ARN/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dorada/genética , Dorada/virología

RESUMEN

Mx proteins are key components of the antiviral state triggered by interferon type I in response to viral infections. In this study, two different Mx genes have been identified in European sea bass (Dicentrarchus labrax), and their sequences were cloned and characterized. MxA cDNA consists of 1881 bp coding for a putative 626 aminoacids protein, while MxB cDNA has 1920 bp and results in a protein with 639 residues. Their corresponding genomic sequences contain 3538 bp and 5326 bp, respectively, and both present 12 exons and 11 introns. The expression patterns of the two Mx genes after an in vivo challenge with the viral nervous necrosis virus (VNNV), a serious pathogen in farmed European sea bass, have been characterized by real-time PCR. The results showed interesting differences in the transcription profile of both Mx, thus suggesting a differential role for each Mx isoform in the immune response of European sea bass to VNNV, and most likely in the general viral response of this species.

Asunto(s)

Lubina/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Unión al GTP/genética , Nodaviridae/inmunología , Infecciones por Virus ARN/veterinaria , Secuencia de Aminoácidos , Animales , Lubina/genética , Proteínas de Unión al GTP/fisiología , Datos de Secuencia Molecular , Proteínas de Resistencia a Mixovirus , Infecciones por Virus ARN/inmunología

RESUMEN

Mx proteins are crucial effectors of the innate antiviral response mediated by the interferon type I signalling pathway. Recently, three Mx proteins, named SauMx1, SauMx2 and SauMx3, corresponding to three different genes, have been identified in the cultured marine species gilthead seabream (Sparus aurata). In this study, the three SauMx cDNAs were cloned into expression vectors and used for transfection of CHSE-214 cells. Monoclonal cell populations stably expressing each recombinant protein have been obtained and characterized. The protection conferred by each recombinant SauMx against Infectious Pancreatic Necrosis Virus (IPNV) infection has been in vitro evaluated, having found clear differences among them. According to the cytopathic effects and the virus yield reduction assays, only cells expressing SauMx2 and SauMx3 showed significant resistance to IPNV infection. Otherwise, quantitative RT real-time PCR assays suggested that each SauMx protein has a different target during the viral inhibition process. The differences observed among the three SauMx proteins are discussed in terms of their differential mechanism of action and antiviral specificity, suggesting, as a whole, to play a synergistic activity in the protection of gilthead seabream against IPNV.

Asunto(s)

Infecciones por Birnaviridae/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Proteínas de Unión al GTP/inmunología , Dorada/inmunología , Animales , Enfermedades de los Peces/virología , Técnica del Anticuerpo Fluorescente , Virus de la Necrosis Pancreática Infecciosa/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

Type I interferons are secreted by infected cells and promote an antiviral state in neighbouring cells by the induction of numerous genes, some of which present antiviral activity, as the Mx proteins. In this study, three different Mx cDNAs (Mx1, Mx2 and Mx3) from gilthead seabream (Sparus aurata), the most important fish species in Southern European aquaculture, have been cloned and characterized. A Southern blot assay revealed the existence of three Mx loci, thus the three Mx isoforms correspond to three different genes that seem to have a common origin. The genomic sequences of Mx1, Mx2 and Mx3 have been completely obtained, and consist on 11 introns and 12 exons in a full length of 5971 bp, 7391 bp and 6938 bp, respectively. As a first approach to the functional meaning of these three genes, their response to the viral nervous necrosis virus (VNNV) infection was tested. Important differences in terms of tissue, time course and level of induction were found between them, thus suggesting a differential functional role for each isoform, which can represent a key point in the natural resistance of this fish species, that has been repeatedly reported as an asymptomatic carrier of VNNV.

Asunto(s)

Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Unión al GTP/genética , Nodaviridae/inmunología , Infecciones por Virus ARN/virología , Dorada/inmunología , Dorada/virología , Secuencia de Aminoácidos , Animales , Southern Blotting , Clonación Molecular , ADN Complementario/genética , Exones/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Genoma/genética , Intrones/genética , Datos de Secuencia Molecular , Proteínas de Resistencia a Mixovirus , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/inmunología , Dorada/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética