Apoptosis Of FHM Cells Induced By Grass Carp Reovirus And The Construction Of Recombinant Baculovirus Expressing VP6Protein

Grass Carp reovirus(GCRV) is the first viral pathogen identified from aquatic in China, which has been considered as one of the most pathogenic agent in the Aquareovirus genus of the Reoviridae family. It can infect not only grass carp but black carp, particularly in fingerling and yearling carp and cause enormous loss in fish industry. Therefore, it is quite urgent to develop the reseach of pathogenesis and an effective vaccine for better prevention and control of GCRV infection. Our study focused on two projects that was to prove that GCRV could induce apoptosis in FHM cells and to construct the recombinant baculovirus-based vaccine against GCRV.1. Preliminary study on GCRV-induced apoptosis in FHM cells CIK and FHM cells were infected by GCRV in this study, and the cytopathic effects were abserved at different times post-infection. It was shown that both of CIK and FHM cells can be infected by GCRV, GCRV infection can cause CIK cells fusion and lysis rapidly. However there was little cell fusions that were observed in infected FHM cells. Apoptosis was detected by DAPI staining and annexin V-FITC staining in infected FHM cells, which was demonstrated that GCRV could induce apoptosis in FHM cells. We considered that GCRV-infected CIK cells may die through non-apoptosis mechanism. Our results demonstrated that GCRV can induce apoptosis in FHM cells and indicated that GCRV may induce different cell types death by different mechanisms, which can help to further understand and study the pathogenesis of GCRV.2. Restructure of the recombinant baculovirus expressing GCRV VP6protein and evaluation of immunity effect of vaccine.The PCR products of VP6gene were inserted into the transfer vector and the recombinant transfer vector had generated, named pFast-CMV-VP6.The transfer vector pFast-CMV-VP6was transformed into E.coli DHlOBac containing baculovirus shuttle vector(bacmid). After isolation and purification, the recombinant bacmid (rBacmid-CMV-VP6) generated was transfected into sf9cells and the recombinant baculovirus (Bac-CMV-VP6) was obtained in sf9cells. It was proved that VP6protein was expressed in Bac-CMV-VP6-mediated sf9, EPC and CIK cells by indirect immunofluorescence assay. The grass carp with10-15cm were intramuscularly immunizated with1x109PFU of recombinant baculoviruses. Afer7d,14d and21post-immunization, liver and spleen tissues were harvested from the fish. The analysis of Real-time quantitative PCR detected the expression of IFN-I and IRF-7genes. In order to examine the level of neutralizing antibody in the serum from vaccinced fish at21days post-immunization, the microneutralization assay was carried out. The result proved that the recombinant baculovirus Bac-CMV-VP6could not only upregulate the expression of IFN-I and IRF-7but elicited the production of neutralizing antibody, which had carved out a novel genetic engineering vaccine based on baculovurise against GCRV.