Generation, characterization and application of monoclonal antibodies against matrix protein of hirame novirhabdovirus (HIRRV) in flounder

Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M piotein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cypiini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the viiusinfected EPC cells, the virions were mainly located in the cytoplasm, wheieas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heait weie higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.