Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell co-receptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4h and a decrease after 24h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.

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In recent years the cloning of genes coding for immuno-regulatory peptides, as well as the sequencing of genomes, provided fish immunologists with a growing amount of information on nucleotide sequences. Research is now also addressed in investigating the functional immunology counterpart of nucleotide sequence transcripts in various fish species. In this respect, studies on functional immunology of T cell activities are still at their beginning, and much work is needed to investigate T cell responses in teleost fish species. In this review we summarise the current knowledge on the group of genes coding for main T cell-related peptides in fish, and the expression levels of these genes in organs and tissues. Particular attention is paid to European sea bass (Dicentrarchus labrax), a marine species in which some information on functional immunology has been obtained, and we reassume here the expression of some T cell-related genes in basal conditions. In addition, we provide original data showing that T cells purified from the intestinal mucosa of sea bass with a specific mAb, express transcripts for TRβ, TRγ, CD8α, and RAG-1, thus showing similarities with intra-epithelial leucocytes of mammals.

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.