Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax).

The gills of fish are a mucosal tissue that contains T cells involved in the recognition of non-self and pathogens, and in this work we describe some features of gill-associated T cells of European sea bass, a marine model species. A whole transcriptome was obtained by deep sequencing of RNA from unstimulated gills that has been analyzed for the presence of T cell-related transcripts. Of the putative expressed sequences identified in the transcriptome, around 30 were related to main functions related to T cells including Th1/Th2/Th17/Treg cell subpopulations, thus suggesting their possible presence in the branchial epithelium. The number of T cells in the gills of sea bass, measured with the specific T cell mAb DLT15 range from 10% to 20%, and IHC analysis shows their abundance and distribution in the epithelium. Leukocytes from gills are able to proliferate in the presence of lectins ConA and PHA, as measured by flow cytometry using CFSE fluorescence incorporation, and during proliferation the number of T cells counted by immunofluorescence increased. In lectin-proliferating cells the expression of T cell-related genes TRß, TRγ, CD4, CD8α, CD45 and IL-10 increased dramatically. Our data represent a first analysis on T cell genes and on basic T cell activities of fish gills, and suggest the presence of functionally active subpopulations of T lymphocytes in this tissue.