Preparation Of Monoclonal Antibody Against Nucleoprotein Of SVCV And Function Of Viperin And Its Splice Variant In SVCV Infection

Spring viremia of carp virus(SVCV)is a negative-sense ss RNA virus and cause high mortality in common carp(Cyprinus carpio),which induces serious aquaculture losses.SVCV encodes five proteins including the nucleoprotein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G),and viral RNA-dependent RNA polymerase(L).N protein is the key protein in nucleocapsid structure.The protein has the highest expression and play an important role in transcription and regulation of virus gene.Mammals Viperin has antiviral functions,and fish Viperins also mediates antiviral activity against several viruses.However,the underlying mechanism remains elusive.In this study SVCV N monoclonal antibody was prepared as an effective tool for study the fuction of SVCV N protein and detecting virus.Meanwhile we explored about what effect might SVCV have on Viperin and its splice variant,along with how Viperin and Viperin_sv1 have influence on SVCV infection.The purpose of the study was revealing the relationship between alternative splicing of Viperin and SVCV infection from the perspective of innate immunity,providing the scientific evidence for analyzing the immunity and pathogenic mechanism and the development of prevention and control products of SVCV.1.Preparation of monoclonal antibody against nucleoprotein of SVCV.Plasmid N-RFP containing Carp sprivivirus as a template,the full-length coding sequence region of SVCV nucleoprotein(N)gene was amplified and cloned into the prokaryotic expression vector p GEX-KG.The fusion protein SVCV-N-KG was immuned BALB/c mice after expressed and purified.The immune B cells of mouse with highest serum titer were fused with SP2/0 cells.One monoclonal antibody(Mc Ab N-2)against N protein were generated and selected by ELISA and chromosome number analysis after four times subcloning selection,The results of IFA(Indirect immunofluorescent assay)and western-blot assay demonstrated the characterizations of Mc Ab N-2.Further analysis showed that Mc Ab N-2 targeted 227~336 amino acid region of N protein.2.Function of Viperin and its splice variant in against SVCV infection.Alternative splicing,a post-transcription regulated process of eukaryotic gene,occurs frequently in immune genes.In this study,we discovered the novel Viperin splice variants named Viperin_sv1 in Spring viremia of carp virus(SVCV)infected Fathead Minnow(FHM)cells.Spring viremia of carp virus(SVCV)was able to induce Viperin and Viperin_sv1 at the m RNA level,while poly(I:C)can induce Viperin but had no notable effect at Viperin_sv1.Viperin and Viperin_sv1 protein partially distributed in the mitochondria and colocalized of the two transcripts was observed in FHM cells.Viperin functions as antiviral protein in the early phase of SVCV infection,but along with SVCV replicating in cells,the antiviral activity will dramatically decline.Viperin_sv1 inhibited virus replication significantly at different time in SVCV infected FHM cells.Unlike mammals,fish Viperin_sv1 but not Viperin was able to induce IFN1 and ISGs including Mxb and PKR expression significantly.Viperin_sv1 increase the expressions of RIG-I,IRF3 and IRF7 significantly,but had no significant effect on NF-?B.The results indicate that fish Viperin_sv1 rather than Viperin induced type I IFN expression by activating IRF3/7 signaling pathway to impairs SVCV replication.Even though Viperin_sv1 expression is highly induced by SVCV,it is negatively regulated at the protein level.Protease inhibitor MG132 protected Viperin_sv1 from protein degradation and rescued its antiviral activity against SVCV.This indicated that SVCV can regulate Viperin_sv1 from protein degradation to counteract its antiviral effect.