Studies On The Apoptosis Induced By Koi Herpesvirus And The Screening Of Anti-virus Drugs

Common carp, including ornamental koi carp, Cyprinus capio L. are ecologically and economically important freshwater fish in Europe and Asia. However, since the Koi herpesviral disease (KHVD) outbrook in1998in Israel and the United States, the Koi herpesviral disease is spreading all over the world. The etiological agent of Koi herpesviral disease has been identified as Koi herpesvirus (KHV), as it is the third herpesvirus isolated from the Cypinid fishes, so it is also known as Cyprinid herpesvirus3,(CyHV-3). Currently, the herpesviral disease caused by KHV infection in common carp represents the most serious infectious disease in China, which has caused huge economic loss. At present, no effective methods are available for the prevention and control of the disease. This study has investigated the apoptosis process induced by KHV in Koi-Fin cells and screened the medicinal herbals in cell culture system for the inhibition of the KHV replication. The results of this study provided a solid basis for the prevention and treatment of the disease in future. The contents and results of the study are as below:1. Apoptosis induced by KHV in Foi-Fin cells. KHV induced apoptosis in Koi-Fin cells was observed by Hoechst33258staining, TUNEL reaction, DNA fragmentation assay, flow cytometric analysis and JC-1assay. KHV caused typical cytopathic effect (CPE) in Koi-Fin cells at96h post-infection by the infection test. A series of charactersistic apoptotic morphological changes were noted by Hoechst33258staining, including chromatin condensation, nuclei marginalization, and apoptotic bodies noted in72h infected Koi-Fin cells, and as the time of KHV infection gone, the apoptotic rate increased. The TUNEL-positive cells proved the Koi-Fin cells genomic DNA was broken. DNA extracted from the KHV infected Koi-Fin cells displayed characteristic180～200bp ladders at48h post-infection and the fragmentation increased with infection time until96h. In the sub-G1cells analysis, the rate of hypo-diploid cells reached42.8%at72h post-infection. Our results suggested that KHV could induced apoptosis in Koi-Fin cells.2. By cytopathic effect (CPE) observation and the staining assay of live cells with MTS, herbal drugs were screened and evaluated for the inhibition of replication of koi herpesvirus (KHV) in Koi-Fin cells. The safety tests of drugs to cells showed that Yinghuang, Astragalus polysaccharide, Radix Isatidis, Herbal Andrographitis and Andrographolide were15.63mg/mL,2.5mg/mL,31.25mg/mL,31.25mg/mL and6.25mg/mL, respectively. In the tested group of drugs added prior to infection, while the concentrations of Yinghuang, Astragalus polysaccharide, Radix Isatidis and Herba Andrographitis were15.63mg/mL,2.5mg/mL,31.25mg/mL and6.25mg/mL, significant inhibition of KHV replication were observed, the treatment index were22.88±3.26,21.07±4.86,24.76±3.24and18.20±2.78, respectively, but the Andrographolide is of no apparent inhibition. In the tested group that the infection was prior to drug addition, at the concentrations of Yinghuang, Radix Isatidis and HerbaAndrographitis of15.63mg/mL,31.25mg/mL and6.25mg/mL, the significant effects in inhibition of KHV replication were achieved, the treatment index were33.21±7.71,21.99±3.12and15.91±3.23, respectively, while the Astragalus polysaccharide and Andrographolide had no apparent effects. In the tested group of co-incubation of virus and drugs before infection, at the concentrations of Radix Isatidis of31.25mg/mL, low efficacy was observed in inhibiting KHV replication, the effect of treatment index were5.7±1.62, while the other drugs had no effects. The results here provided a solid basis for the application of drugs in preventing and controlling the common carp herpesviral disease in farming practice.