The epizootic haematopoietic necrosis virus (EHNV) is an iridovirus causing severe disease in different fish species. We investigated the induction of apoptosis during EHNV infection of the epithelioma carp papulosum (EPC) cell line. Apoptosis reveals several characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, cytoplasm membrane disorientation, or mitochondrial changes. During EHNV infection of EPC cells the occurrence of apoptosis was analysed using a fluorescein-isothiocyanate (FITC) conjugate of annexin-V to detect phosphatidylserines that have changed cytoplasm membrane localization. Annexin-V labelling was obvious 12 h after infection. At 54 h after EHNV infection 39% of the investigated EPC cells exhibited fluorescence. Furthermore, EHNV-infected cells were stained with 4'-6'-diamidino-2-phenylindole (DAPI) to detect pycnotic nuclei. Appearance of DAPI-positive nuclei was found beginning at 18 h after infection. At 54 h after EHNV infection approximately 56% of the EPC cells showed fragmented nuclei. Assays to inhibit a protein kinase-dependent (e.g. double-stranded RNA-dependent protein kinase) apoptosis pathway with 2-aminopurine revealed a reduction of EHNV titres, e.g. titres were reduced 1000-fold in the presence of 100 and 200 mM 2-aminopurine. Apoptosis takes place during iridovirus infection in vitro and it seems to involve the activation of protein kinases.
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