|Other Abstract||Cell culture and monoclonal antibodies (mAbs) techniques are still now basic approaches on the virological investigation. Though it has a solid research foundation on the establishment systems of cell lines and mAbs for medical virology, much more information on the above two aspects need to be accumulated in lower vertebrate animals (i.g. the fish) virology research. In addition, exploring the functional genes of aquatic viruses could provide some fundamental knowledge for the control of virus infection. In this thesis, combining the fish rhabdoviruses, one of the most important viral pathogens in aquaculture, three major work involved in the establishment of fish new cell lines, virus mAbs production and identification of virus gene were done. The main research results are given as follows:
1. Establishment of two new fish cell lines and their preliminary application. Grouper snout (GS) and Chinese sturgeon tail fin (CSTF) cell lines were successfully established and characterized from healthy red spotted grouper and Chinese sturgeon, respectively. Morphologically, the GS cells were fibroblastic, while the latter consists of epithelial-like polygonal cells. Both of them could be maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% fetal bovine serum (FBS) for more than 60 subcultures and the highest growth rate was obtained at 25℃. With free-FBS medium, GS cells could not grow well while trypsinized CSTF cells could not adhere to the flask substratum. Karyotypic analysis revealed that both of the two cell lines keep a normal diploid karyotype, with the chromosome modal number 48 (GS) and 264 (CSTF), respectively. The CSTF cells contain large numbers of punctate chromosomes. Two strains of aquatic animal viruses (Scophthalmus maximus rhabdovirus, SMRV; Rana grylio virus, RGV) were used to test the
susceptibility of the GS and CSTF cells to infection. SMRV could infect the GS cell line but not produce cytopathic effect (CPE) in CSTF cells. The two cell lines both show sensitivity to RGV. Infection was also confirmed by immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of virus-infected cells. Furthermore, after transfection with pEGFP vector DNA, the transfected GS and CSTF cells all produced significant fluorescent signals indicating their potential utility in exogenous studies.
2. Production and characterization of monoclonal antibodies (mAbs) against SMRV or PORV (Paralichthys olivaceus rhabdovirus). The SMRV and PORV antigens were purified with differential centrifugation followed by sucrose density gradient centrifugation from virus-infected fish cells, respectively. Purified virus antigens were used to immunize intraperitoneally Balb/c mice. After fusion, screening and limiting dilution, five mAbs (1G8, 1H9, 2D2, 2D3 and 2F5) specific to SMRV and two mAbs (3C7 and 3C9) against PORV were finally isolated and cloned. Isotype determination showed that the 1G8 and 1H9 belong to IgG2b, while the others are IgM subclass. Western blotting analysis showed that all the 5 SMRV mAbs could react with two viral protein bands of 30 and 26 kDa, while 3C7 and 3C9 reacted with three PORV virus proteins (42 kDa, 30 kDa and 22 kDa). All the mAbs produced staining signals in the cytoplasm of virus-infected EPC or GCO cells. 2D3 and 3C7 were also used to detect the viral antigens in SMRV-infected flounders and PORV-infected carp tissues respectively, which showed positive immunostaining in tissue sections. Furthermore, flow cytometry was applied for detection the virus-infected fish cells with the above two mAbs. The results suggested that 36 h postinfection, the SMRV-infected epithelioma papulosum cyprinid (EPC) (0.01PFU/cell) and PORV-infected grass carp ovary (GCO) (0.25PFU/cell) cell populations could be distinguished from uninfected cells, respectively. Approximately 10.8% of EPC cells were infected with SMRV, while the percentage of PORV-infected GCO cells came to 23%.
3. Inhibition of Siniperca chuatsi rhabdovirus (SCRV) by RNA interference (RNAi) and its specific cleavage of the nucleoprotein in a fish cell line. Several small interfering RNAs (siRNAs) corresponding to nucleoprotein (N) gene of the SCRV were chemically synthesized to explore the possibility of silencing the gene and inhibition the virus replication
in EPC cells. Cytopathic effect observation and Western blotting analysis both suggested apparent inhibition effect of N gene expression and virus replication by si983, which presented a dose dependent activity.
Furthermore, immunoblotting also showed that except to the full-length N protein band, two additional bands (41 kDa and 35 kDa) were detected in SCRV-infected or N-GFP transfected EPC cell line, which had not been recorded before in rhabdoviridae. The caspase-3 activity in infected or transfected EPC cells was higher five times or twice than controls’. When treated with z-VAD-FMK (100 μM, a caspases pan-inhibitor) in the above cells, the cleavage of N protein became fainter, even disappeared. But, treatment of z-VAD-FMK could not reduce the progeny virus production in SCRV-infected cells. Combined with the results of caspase-3 activity assay and z-VAD-FMK inhibition experiments, we indicated that the lower bands of N were the results of caspase-mediated cleavage. To further verify the proteolytically cleavage sites, several kinds of nucleoprotein gene truncated mutant fragments (N1-GFP [aa1-376], C1-GFP [aa377-429], N2-GFP [aa1-281], C2-GFP [aa282-429], C3-GFP [aa325-429]) were constructed and transfected transiently into EPC cells for immunoblotting analysis. According to the differences of every truncated protein fragments molecular weights and cleavage products in transfected cells, processing of N at residues D324 and D374 by caspases was finally identified, which is the first report on the cleavage of rhabdoviruses nucleoproteins.|