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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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.)

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., (Copyright © 2012 Elsevier Ltd. 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.