Studies on the apoptosis of channel catfish(Ictalurus punetaus) kidney cells induced by channel catfish reovirus

Abstract

Channel catfish reovirus(CCRV)is the pathogen of the hemorrhage of channel catfish(Ictalurus punctatus Rafinesque),which propagates in the kidney of channel catfish(CCK)cell line.In this study,the evaluation of whether the CCRV could induce the apoptosis of CCK cells was examined by Hoechst33258 staining,DNA fragmentation assay,TUNEL reaction,and flow cytometry analysis using JC-1,etc.The infection test indicated that CCRV caused typical cytopathic effect(CPE)in CCK cells.Chromatin condensation,nuclei marginalization and the apoptotic bodies were observed in Hoechst 33258 staining,and the apoptotic rate increased with the time going of CCRV infection.The DNA fragmentation assay demonstrated that fragmentation was first noted at 12 h post-infection and reached the peak at 72 h post-infection.In addition,genomic DNA was broken and lots of 3′-termianl free-hydroxyl group(-OH)were generated in infected CCK in TUNEL assay.The hypo-diploid fraction was shown in the sub-G1 cells analysis and the apoptotic rate was 53.44% at 48 h post-infection.Furthermore,the change of the mitochondrial membrane potential(MMP)was investigated by flow cytometric analysis with JC-1 fluorescent labeling and the result showed that the membrane permeability and MMP of CCK cells changed significantly at 24 h post-infection.These results demonstrated that CCRV induced apoptosis in CCK cells.Besides,CCRV-induced apoptosis did require the viral replication as the apoptosis was blocked in CCK cells by both heat-inactivated and UV-inactivated virus.