Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody

An enzyme-immunohistochemical procedure was employed to facilitate neutralization/diagnostic tests for viral haemorrhagic septicaemia virus (VHSV), a significant pathogen in trout farms throughout Europe. The method described can be used for trout or mice antibodies; increases speed (1 day), simplicity, and minimizes the use of reagents compared to other neutralization assays. Furthermore, the test requires a minimum handling of the cell cultures under sterile conditions, decreasing frequent contamination due to the non-sterile conditions of the fish pathological samples. Foci of 5-20 infected epithelioma papillosum carp (EPC) cells are detected and counted with an inverted micrcoscope in under 16 h after infection of EPC monolayers using a high titre anti-N VHSV monoclonal antibody (MAb) 2C9. MAb 2C9 recognizes different viral haemorrhagic septicaemia virus serotypes and VHSV isolates from different host species (trout, salmon and barbel) and Spanish geographical locations. The high titre and specificity of MAb 2C9 favour its conjugation to peroxidase and also make it possible to use in direct immunoperoxidase staining of the VHSV infected EPC monolayers. This neutralization/immunoperoxidase assay should improve diagnostics hat use currently agarose or methylcellulose plaque reduction neutralization assays.