Preliminary Study On Relationship Between Apoptosis And Proliferation Induced By Spring Viremia Of Carp Virus

Spring viremia of carp virus(SVCV)is the pathogen of Spring viremia of carp(SVC).SVC of cyprinidae fish breeding great harm,and its pathogenesis is unclear,the international organization for animal health(OIE)will SVC as one of the fish disease must be reported,and the Ministry of Agriculture of the People’s Republic of China regards the SVC as the first grade Animal disease.Apoptosis is a cellular response to various stimuli active,highly ordered process of programmed cell death,a stable of great significance to maintain the body’s internal and external environment.In order to understand the pathogenesis of SVCV,this study preliminarily explored the proliferation of SVCV virus in cells and the mechanism of SVCV induced apoptosis.First,culturing EPC and CO after challenge and comparing results of TCID50,the best sensitive cells were selected for follow-up experiments;by establishing the SVCV-G protein standard curve,qRT-PCR method was used to observe SVCV’s sensitive cells at different times and explore,the relationship between the proliferation of the virus and the cell disease.The results showed that EPC cells were more sensitive to SVCV than CO cells.The result of RT-PCR detection showed a significant increase in virus volume after 8h culture,reached a peak at 64 h,and was consistent with the obvious pathological changes of 16-32 h cells when they were observed in the form of anti-virus cells.Second,using Hoechst33258 and transmission electron microscopy(TEM)to observe the morphogenesis of the cell cultured for 64 h after challenge,preliminary validation of SVCV induced apoptosis.Apoptosis was detected by flow cytometry using Annexin V-FITC/PI double staining and the expression levels of caspase-3,8 and 9 were detected by inhibitors at different time points.Using CytoTox 96 non-radioactive cytotoxic detection method,the concentration of LDH in the post-epc culture medium infected with SVCV was detected,which indirectly reflected the cell viability.The results showed that Hoechst 33258 staining revealed that the EPC cells infected with SVCV exhibited dense staining of nuclei and strong nuclear fluorescence,the transmission electron microscopy showed that the chromatin aggregated to the edge and gradually detached from the cytoplasm and eventually formed apoptotic bodies,the series of cell apoptosis typical characterization,confirmed that SVCV can induce apoptosis in EPC cells;The expression of caspase-3 increased with the time of virus action,peaked at 8 h,and then decreased gradually.The expression of caspase-8 increased with time,and reached the highest level at 8h,after which the expression level decreased.The change of caspase-9 expression was similar to that of caspase-3 and 8,there was time compliance,and the expression of caspase-9 was the highest at 16h;When the CytoTox 96 non-radioactive cytotoxicity assays culture 8h,Compared with non-challenge groups,the concentration of LDH significantly increased,for 16 h after culturing,in particular 32 h,64h,LDH infection is much higher.The experiment initially explored the impact of SVCV on EPC cells.The virus can induce EPC apoptosis;the determination of the virus proliferation and apoptosis determine the active period of cells.According to the increase of expression of caspase-3,8 and 9,it is inferred that there are two apoptotic pathways in SVCV induced apoptosis.This research lays the foundation for the mechanism of SVCV-induced apoptosis in EPC cells,which will provide a direction for the further study of the pathogenic mechanism of SVCV,and a theoretical basis for the treatment,medication and prevention and control of SVC.