Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines.

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.

Alvarez-Torres D ，Alonso MC ，Garcia-Rosado E ，Collet B ，Béjar J ... -  《-》

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 ... -  《-》

Structural and functional characterization of the Senegalese sole (Solea senegalensis) Mx promoter.

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.

Alvarez-Torres D ，Bejar J ，Collet B ，Alonso MC ，Garcia-Rosado E ... -  《-》

Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages.

Collet B ，Munro ES ，Gahlawat S ，Acosta F ，Garcia J ，Roemelt C ，Zou J ，Secombes CJ ，Ellis AE ... -  《FISH & SHELLFISH IMMUNOLOGY》

In vivo virulence of viral haemorrhagic septicaemia virus (VHSV) in rainbow trout Oncorhynchus mykiss correlates inversely with in vitro Mx gene expression.

The in vitro replication of viral haemorrhagic septicaemia virus (VHSV) isolates from each VHSV genotype and the associated cellular host Mx gene expression were analysed. All the isolates were able to infect RTG-2 cells and induce increased Mx gene expression (generic assay detecting isoforms 1 and 3 [Mx1/3]). A trout pathogenic, genotype Ia isolate (J167), showing high replication in RTG-2 cells (by infective titre and N gene expression) induced lower Mx1/3 gene expression than observed in VHSV isolates known to be non-pathogenic to rainbow trout: 96-43/8, 96-43/10 (Ib); 1p49, 1p53 (II); and MI03 (IVb). Paired co-inoculation assays were analysed using equal number of plaque forming units per ml (PFU) of J167 (Ia genotype) with other less pathogenic VHSV genotypes. In these co-inoculations, the Mx1/3 gene expression was significantly lower than for the non-pathogenic isolate alone. Of the three rainbow trout Mx isoforms, J167 did not induce Mx1 up-regulation in RTG-2 or RTgill-W1 cells. Co-inoculating isolates resulted in greater inhibition of Mx in both rainbow trout cell lines studied. Up-regulation of sea bream Mx in SAF-1 cells induced by 96-43/8 was also lower in co-inoculation assays with J167. The RTG-P1 cell line, expressing luciferase under the control of the interferon-induced Mx rainbow trout gene promoter, showed low luciferase activity when inoculated with pathogenic strains: J167, DK-5131 (Ic), NO-A-163/68 (Id), TR-206239-1, TR-22207111 (Ie), 99-292 (IVa), and CA-NB00-01 (IVc). Co-inoculation assays showed a J167-dose dependent inhibition of the luciferase activity. The data suggest that virulent VHSV isolates may interfere in the interferon pathways, potentially determining higher pathogenicity.

Cano I ，Collet B ，Pereira C ，Paley R ，Aerle RV ，Stone D ，Taylor NGH ... -  《-》