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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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

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