Studies On The Apoptosis Of Channel Catfish (Ictalurus Punetaus)Kidney Cells Induced By Channel Catfish Reovirus

The disease of Channel catfish (Ictalurus punctatus Rafinesque) hemorrhage was broke out in a farm of culturing the fish in Hube Province. It was characterized by hemorrhages of gills, operculum, skin, fin bases, and abdominal swelling, malformation, etc. This disease mainly affected the channel catfish fingerlings; the mortality of diseased fish was as high as60%and caused considerable economic losses. Channel catfish reovirus is the causative pathogen of the disease, which belongs to the genus of Aquareovirus.In this study, the method for culturing CCRV in channel catfish kidney cell line (CCK) was established. CCRV could induce the typical cytopathic effect (CPE) in CCK cell line and the virus titer could reach10-867/ml. By using microculture system to titrate the virus titer after frozen-thawed treatment, the result showed that frozen-thawed treatment had no significant effect on the virus titer.Apoptosis is a contributing element for virus pathogenicity, it is essential to determine whether the major mechanism of cell death occurring during the virus infection in vitro is associated with the induction of apoptosis. In this study, the apoptosis induced by CCRV in CCK cells was examined by Hoechst33258staining, DNA fragmentation assay, TUNEL reaction, and flow cytometric 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 Hoechst33258staining, and the apoptotic rate increased with the time going of CCRV infection. The DNA fragmentation assay demonstrated that fragmentation was first noted at12h post-infection and reached the peak at72h post-infection. In addition, genomic DNA was broken and lots of3’-termianl free-hydroxyl group (-OH) was generated in infected CCK in TUNEL assay. The hypo-diploid fraction was shown in the sub-G1cells analysis and the apoptotic rate was53.44%at48h post-infection. Furthermore, the change of the mitochondrial membrane potential (MMP) was investigated by flow cytometric analysis with JC-1fluorescent labeling and the result showed that the membrane permeability and MMP of CCK cells changed significantly at24h post-infection. These above 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.