Cell-mediated citotoxicity of fish leucocytes against betanodavirus

Résumé

Farmed fish are subjected to great stress, affecting their immune system and making them vulnerable to disease. Among the most lethal pathogens are viruses, most notably Viral Nervous Necrosis Virus (VNNV), which causes viral encephalopathy and retinopathy disease (VER). In the Mediterranean Sea, the RGNNV genotype is mainly prevalent, being able to infect European sea bass and gilthead seabream, the latter being resistant to VER. However, the appearance of recombinant genotypes (RGNNV/SJNNNV and SJNNNV/RGNNV) showed that seabream was capable of developing the disease. Among the mechanisms presented by teleost fish against viral infections, cell-mediated cytotoxicity (CMC) is the most important, carried out by different cell types, with non-specific cytotoxic cells (NCCs) and natural killer-like cells (NK-like) in the innate system, while in the adaptive system it is carried out by cytotoxic T-lymphocytes (CTLs). Seabream is able to mount an effective innate response against NNV, highlighted by their innate CMC, capable of eliminating infected cells, whereas sea bass is not. Therefore, the aim of this Doctoral Thesis is to deepen in the dynamics of the leukocyte-mediated cytotoxic response of sea bass and seabream against NNV. For this purpose, Chapter I studied the interaction of recombinant genotypes of NNV in seabream larvae of different ages, evaluating mortality and genes involved in the infections. Results showed that both genotypes could infect larvae of different ages, with higher pathogenicity in younger larvae. The RGNNV/SJNNV genotype produced higher mortality due to higher viral prevalence and lower activation of the immune system. Chapter II characterised the CRTAM receptor (present in mammalian T lymphocytes) and its ligand CADM1 in both species by bioinformatic analysis and evaluated their gene expression under different conditions, including NNV infection. The analysis identified one crtam gene and two cadm1 genes (cadm1a and cadm1b) in both species. The crtam gene was mainly expressed in immune tissues, while cadm1a and cadm1b were mostly expressed in brain and skin. In sea bass, crtam appears to be expressed in T lymphocytes and to be involved in NNV infection, while in seabream it is expressed in NCCs or NK-like. Chapter III focuses on the identification of perforin in sea bass by bioinformatic analysis and the study of its gene expression in different conditions as well as in an NNV infection, in addition to assessing the presence of perforin-producing cells by immunohistochemistry in an NNV infection. Four different perforin genes, prf1.2, prf1.3, prf1.5 and prf1.9, were found, all showing basal expression in different tissues and synthesised mainly by T lymphocytes, except prf1.3. Furthermore, all genes, except prf1.3, were involved in the infection against NNV, supported by the generation of PRF1.9-producing cells in brain, cephalic kidney and gonad. Finally, in Chapter IV, adaptive CMC was assessed using NNV-infected sea bass leukocytes as effector cells and DLB-1 cells (from sea bass brain) as target line. Innate cytotoxicity did not increase during infection, but the cytotoxicity of leukocytes from infected fish did increase in late stages of infection, indicating activation of the adaptive immune system. This increase was specific against genotypes sharing the same viral capsid, supported by a possible MHC-I restriction. In this adaptive cytotoxicity, an increase in perforin/granzyme and Fas/FasL pathways, characteristic of CTLs, was observed, as well as an increase in apoptosis.