Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

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.

Álvarez-Torres D ，Gómez-Abellán V ，Arizcun M ，García-Rosado E ，Béjar J ，Sepulcre MP ... -  《-》

Mx1, Mx2 and Mx3 proteins from the gilthead seabream (Sparus aurata) show in vitro antiviral activity against RNA and DNA viruses.

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.

Fernández-Trujillo MA ，García-Rosado E ，Alonso MC ，Castro D ，Álvarez MC ，Béjar J ... -  《-》

Expression pattern, antiviral role and regulation analysis of interferon-stimulated gene 15 in black seabream, Acanthopagrus schlegelii.

Interferon stimulated gene 15 (ISG15) is an IFN inducible ubiquitin-like protein and plays a critical role in immune response against viral infection. In this study, an ISG15 gene (AsISG15) was cloned and characterized from the marine fish black seabream, Acanthopagrus schlegelii. The full-length cDNA of AsISG15 was 1302 bp and encoded 155 amino acids containing two ubiquitin-like motifs and a LRGG conjugation domain. Multiple alignment and phylogenetic tree showed that AsISG15 shared 31-70% amino acid identity with other known ISG15s and had a closer evolutionary relationship with teleost ISG15s. In vitro, AsISG15 expression was inducible by poly I:C, LPS and red spotted nervous necrosis virus (RGNNV) in cultured black seabream brain cells. In vivo, AsISG15 was ubiquitously expressed in all examined tissues with higher expression levels in eye and gill, and the expression was significantly up-regulated in most tissues post RGNNV infection, especially in liver, spleen and kidney. The testing of antiviral activity showed that silencing AsISG15 significantly increased RGNNV replication in RGNNV infected AsS cells, and the LRGG domain was crucial for the anti-RGNNV activity of AsISG15. By promoter-driven luciferase reporter assay, we demonstrated that two IFN-stimulated response elements within the promoter region of AsISG15 and the promoter-proximal intron were essential for AsISG15 expression. Furthermore, our results showed that the gamma-IFN activation sequence located in the intron was required for the intron mediated enhancement for AsISG15 expression. Our results would provide insights for understanding the underlying regulation mechanism of ISG15 in teleost.

Liu W ，Xiang Y ，Zhang W ，Jia P ，Yi M ，Jia K ... -  《-》

Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells.

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.

González-Mariscal JA ，Fernández-Trujillo MA ，Alonso MC ，García-Rosado E ，Álvarez MC ，Béjar J ... -  《-》

Molecular characterization and expression analysis of interferon-gamma in black seabream Acanthopagrus schlegelii.

Interferon gamma (IFN-γ) is a major component in immunological signaling and plays a key role in resisting viral infection. In this study, we identified and characterized an IFN-γ gene (AsIFN-γ) in the marine fish black seabream (Acanthopagrus schlegelii). We cloned AsIFN-γ genomic sequence, which comprises four exons, three introns and an upstream promoter including several conserved regulatory elements. The complete cDNA of AsIFN-γ was 816 bp in length and encoded a putative 194 amino acids (aa) protein with a 22 aa signal peptide, six α-helices and one nuclear localization signal (NLS). Multiple alignment showed that AsIFN-γ protein shared 31-60% identity with IFN-γ of other fish but low identity with fish IFN-γrel and IFN-γ of other vertebrates. AsIFN-γ was constitutively expressed in all examined tissues with the highest expression level in immune organs, such as spleen, gill and kidney. In black seabream infected by red spotted nervous necrosis virus (RGNNV), the expression of AsIFN-γ was significantly up-regulated in most tissues, and RGNNV infection in vitro also induced significant up-regulation of AsIFN-γ, indicating that AsIFN-γ was involved in immune response to RGNNV infection. Overexpression of AsIFN-γ in cultured Acanthopagrus schlegelii brain (AsB) cells rapidly and transiently stimulated the expression of JAK-STAT signaling pathway related genes including STAT1, STAT2 and IRF9, as well as the downstream antiviral genes MX1 and ISG15. Furthermore, overexpression of AsIFN-γ was able to significantly inhibit RGNNV replication and virus production in AsB cells. In summary, we identified a conserved IFN-γ gene of black seabream, and demonstrated the rapid and strong antiviral activities of AsIFN-γ against RGNNV in black seabream.

Xiang Y ，Liu W ，Jia P ，Li Y ，Jin Y ，Chen L ，Zhang J ，Jia K ，Yi M ... -  《-》