Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Upregulation of Galectin-1 Expression in Head Kidney, with Potential Anti-Inflammatory Activity

Sea bass nervous necrosis virus (SBNNV) is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. An 8.7 % of the ESTs found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two Ctype lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo upregulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were carried out. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1β, TNF-α and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared to those infected with nodavirus alone. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection. Introduction Sea bass nervous necrosis virus (SBNNV) is a non-enveloped, positive stranded RNA virus that belongs to the Betanodavirus genus, within the Nodaviridae family, and the causative agent of the viral nervous necrosis (VNN). This disease, reported in a wide range of teleost fish, is characterized by lethargy, abnormal behavior, loss of equilibrium and spiral swimming and, histopathologically, by the development of vacuoles in cells from the nervous system tissues (brain, retina and spinal cord) leading to a high mortality in larval and juvenile stages of affected fish (1-3). The mechanism(s) of SBNNV infectivity and pathogenesis are largely unknown and it remains to be determined whether the virus evades, actively blocks, or uses the molecular recognition factors of the host’s immune system. The innate immune response is the first line of defense against microbial pathogens, and consists of a complex network of diverse humoral and cellular recognition and effector components, whose tight regulation contributes to activate or deactivate specific anti-microbial pathways (4). Among these components, lectins are carbohydrate-binding proteins involved in pathogen recognition and neutralization that can also trigger effector mechanisms leading to pathogen destruction and instruction of the adaptive immune mechanisms (5-8). Most animal lectins can be classified into distinct families characterized by unique sequence motifs and structural folds: S-type lectins (galectins), C-type lectins (including selectins, collectins and hyalectans), F-type lectins, I-type lectins, P-type lectins, pentraxins, ficolins, and cytokine lectins (9, 10; see also www.imperial.ac.uk/research/animallectins/). Although lectins from the various families differ greatly in the domain architecture, they are all characterized by a carbohydrate-recognition domain (CRD), which confers the protein its carbohydratebinding activity (11). Galectins are Ca independent soluble lectins, widely distributed in vertebrates, invertebrates (sponges, worms, and insects), and protists (protozoa and fungi) (5, 10, 12, 13), characterized by their affinity for β-galactosides (5, 12, 14). The mammalian galectins have been structurally classified into proto-, chimeraand tandemrepeat types (15). The proto-type galectins exhibit a single CRD per subunit, and in most cases form non-covalently bound dimers. In addition to the CRD at the Cterminus, the chimera-type galectins have an N-terminus region consisting in a short domain followed by an intervening proline/glycine/tyrosine-rich domain with short repetitive sequences. Tandem-repeat-type galectins consist of two homologous CRDs tandemly arrayed in a single polypeptide, joined by a short glycine-rich region (9, 15). Further, a novel tandem-repeat type galectin with four CRDs has been recently described (16). In contrast, C-type lectins, a group of highly diversified soluble or integral membrane lectins, require Ca for ligand binding and their characteristic CRD (C-type-like domain, CTLD) can be classified into those that bind mannose and fucose, and those that bind galactose (8). Members of the pentraxin lectin family, such as Creactive protein and serum amyloids are characterized by subunits arranged in a cyclic oligomeric structure. In some species, including man, pentraxins are expressed upon stimulus by inflammatory cytokines and are considered critical components of the acute-phase response (17). Little is known, however, about the potential role(s) of lectins in the innate immune and tissue repair responses to viral infection in fish. In this study, we used suppression subtractive hybridization (SSH) to examine the sea bass (Dicentrarchus labrax) gene expression profiles in head kidney upon nodavirus infection. A galectin, two C-type lectins, and a pentraxin were identified among the upregulated genes, and were characterized in further detail. Moreover, evidence about the potential anti-inflammatory and protective role of the sea bass galectin on nervous tissue affected by viral infection was obtained. Materials and Methods Fish and virus Adult sea bass (80 g each) were obtained from a commercial fish farm. Prior to the experiments, fish were acclimatized to laboratory conditions for 3 weeks, maintained at 20 oC and fed daily with a commercial diet. The nodavirus strain 475-9/99 isolated from diseased sea bass was provided by the Istituto Zooprofilattico Sperimentale delle Venezie (Italy). Nodavirus challenge and RNA isolation Fish care and the viral challenge experiments were reviewed and approved by the CSIC National Committee on Bioethics. Fish were injected intramuscularly with 100 μl of nodavirus suspension in Minimum Essential Medium (MEM) (10 TCID50 ml ) and placed at 25 oC. Mock-infected control fish (n = 12) were injected with medium alone, and maintained under the same experimental conditions. Three fish from each experimental and control groups were sampled 4, 24, 48 and 72 hours post-infection. Animals were sacrificed by anesthetic (MS-222) overdose, dissected, and the head kidney collected. RNA from individual organs was isolated by homogenization with Trizol reagent (Invitrogen) following the manufacturer’s protocol. RNA was treated with Turbo-DNA free kit (Ambion) to remove contaminating DNA. SSH library construction The SSH technique (18) was used to identify genes in head kidney that may be upregulated upon viral infection. Because preliminary RT-PCR experiments had shown up-regulation of several immune genes 4 hours after viral challenge (data not shown), this timepoint was selected for sampling specimens dedicated to the SSH library construction. cDNA was synthesized from 1 μg of head kidney (infected and control) RNA using the SMART PCR cDNA Synthesis Kit (Clontech). The SSH library was constructed using the PCR-select cDNA Subtraction Kit (Clontech). The PCR products from the forward subtraction procedure were cloned using the Original TOPO T/A Cloning Kit (Invitrogen), and screened by PCR using specific primers. Sequence analysis Excess of primers and nucleotides was removed by enzymatic digestion using 10 and 1 U of ExoI and SAP, respectively (Amershan Biosciences) at 37 oC for 1h followed by inactivation of the enzymes at 80 oC for 15 min. The PCR products were sequenced in a 3730 DNA Analyzer (Applied Biosystems). Blastn and Blastx searching from the NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) were used to identify homologous nucleotide and protein sequences. Protein sequence alignments were evaluated using ClustalW program (http://align.genome.jp/) and specific conserved domains were searched with several programs: Blastp from NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), PROSITE (http://www.expasy.ch/prosite/) and SMART (http://smart.embl-heidelberg.de/). Compute pI/Mw (http://www.expasy.ch/tools/pi_tool.html) for Mw detection; Signal IP (http://www.cbs.dtu.dk/services/SignalP) for signal peptide detection; TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and SOSUI (http://bp.nuap.nagoyau.ac.jp/sosui/sosui_submit.html) for transmembrane domain detection and Disulfind (http://disulfind.dsi.unifi.it) for disulfide bonds were also used.