In the present study, we aims to prevention and control Scophthalmus maximus rhabdovirus (SMRV) and Rana grylio virus (RGV), using flounder Paralichthys olivaceus, grouper Epinephelus akaara and grass carp Ctenopharyngodon idellus as the experimental models to study fish antiviral immune response, respectively. The main results were described as follows:
Firstly, in this study, the full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one α-helix, 13 transmembrane -strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis.
Secondly, this study determined whether specific antibodies were present in the excised skin explants of grass carp, immune to SMRV and RGV viruses, respectively. The antiserum and culture fluid from immune skin explants are detected by iELISA, western blot, IFA and flow cytometry, respectively. The results of iELISA showed that cutaneous antibody titer levels were much lower (1:12) than antiserum titer (1:1458) from intraperitoneal (IP) immunized grass carp. The phosphoprotein (P) and matrix protein (M) antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected EPC cells by IFA technique. Results of flow cytometry showed that 4.39% SMRV-infected EPC cells were detected, while control cells only come to 2.0% in view of the unspecific reaction.
In addition, following injection with RGV virus, specific antibody levels of immunised fish skin culture supernatant were determined by iELISA. Specific antibody can be detected in immunised fish skin culture supernatant. The immunised grass carp serum IgM heavy chain （88 kDa）and light chain（21 kDa） proteins were recognized by rabbit anti-fish antibody using western blot. The skin culture fluid produced staining signals in the perinuclear region of cytoplasm of RGV-infected EPC cells by IFA technique, indicating the viromatrix of RGV distributed in the perinuclear region of cytoplasm. The above findings indicated that skin mucosal immunity play important roles in fish against virus infection.
Thirdly, to further extend the above findings, skin culture supernatant from non-immunized grouper Epinephelus akaara collected and tested for antiviral activity against RGV and SMRV viruses using cell-based assays, respectively. The results demonstrated that fish skin supernatant fluids have non-specific antiviral activities against RGV and SMRV, respectively. Heat-treated supernatant fluids (56℃, 30 min) displayed both the anti-RGV and anti-SMRV activities, indicating that were not heat labile and thus probably not complement mediated.