Development Of Vector Vaccine And CircRNA Vaccine Anti Infectious Spleen And Kidney Necrosis Virus In Fish

The infectious spleen and kidney necrosis virus(ISKNV),belonging to the genus megalovirus,can infect the Siniperca chuatsi and Micropterus Salmoides with very high lethality.Therefore,it is a virulent virus that seriously endangers the development of the aquaculture industry of Siniperca chuatsi and Micropterus Salmoides.Production of an efficient,safe and convenient novel vaccine is urgently needed to combat ISKNV.1.Evaluation of immune effect of live vector vaccine against ISKNVIn this study,based on the baculovirus expression system,the DNA sequence of the major capsid protein(MCP)of ISKNV was selected and controlled by CMV promoter,then cloned into the baculovirus expression vector pFASTTMDual to construct the DNA vaccine pFASTMDual-CMV-MCP of ISKNV.This recombinant baculovirus was constructed by Bac-to-Bac system,named as BmNPV-MCP,which was inoculated into EPC cells from fishes.The results of qPCR,immunofluorescence showed that BmNPV-MCP could enter EPC cells and express MCP protein.Siniperca chuatsi(length 10-15 cm,weight 40-60 g),Micropterus Salmoides(length 8-12 cm,weight 35-55 g)were injected with 8×109 copies of recombinant BmNPV-MCP per fish.Tissues were extracted after inoculation for one week and detected by PCR and qPCR that the recombinant virus was able to enter the kidney and spleen and express vaccine proteins;After 1,2,3 and 4 weeks of immunization,the spleen and kidney tissues were taken for qPCR detection and the kidney tissues kidney immunized for 3 weeks were taken for immunohistochemistry detection.These results demonstrated that BmNPV-MCP could enter spleen and kidney tissues of perches and express MCP.The expression levels of immune related genes of the spleen and kidney tissues of perches immunized with BmNPV MCP for 3 weeks were detected by qPCR.The results showed that the expression of transforming growth factor(TGF-β),tumor necrosis factor α(TNF-α),interleukin-8(IL-8),immunoglobulin M(IgM)and interleukin-1(IL-1)were significantly increased.ELISA results showed that antibody levels reached 1:12800 at three weeks of immunization with BmNPV-MCP.After Four weeks of immunization,ISKNV was inoculated for immune protection test.The results showed that after artificial infection with ISKNV,the relative immune protection rate of perches immunized with BmNPV-MCP reached 87.1%.Perches(length 8-12 cm,weight 35-55 g)was immunized by immersion in BmNPV MCP recombinant virus solution(8 × 1011 copies)for 3 h,and inoculated with ISKNV after four weeks of immunization.The results showed that the protective rate of immersion immunization of BmNPV-MCP was approximately 61.3%,indicating that BmNPV-MCP could be inoculated into fish by immersion and could prevent the invasion of ISKNV.These results indicate that BmNPV-MCP can be used as DNA vaccine for the prevention and treatment against ISKNV.2.Initial assessment of immune efficacy of anti-ISKNV CircRNA vaccinemRNA vaccine is a new direction of vaccine research and development.However,due to the poor stability of mRNA vaccine,it needs cryopreservation and cold chain transportation,which is the challenge that mRNA vaccine must face.Circular RNA is a covalently closed circular single stranded RNA,which is stable in structure and not easy to be degraded by RNase R and other exonucleases.It can initiate protein translation through an Internal ribosome entry site(IRES)independent of cap structure.N6 methyladenosine(m6A)can recruit ribosomes to promote translation.Therefore,circRNA has the potential to be developed as a vaccine.To develop a circRNA vaccine against ISKNV,a recombinant expression vector pFASTMDual-IRES-m6A-MCP with T7 promoter and MCP coding sequence controlled by encephalomyocarditis virus internal ribosomal entry site(IRES)and m6A site sequence(T7-IRES-m6A-MCP)was constructed manually.T7-IRES-m6A-MCP fragment was obtained by BamH I/XBA conjugate digestion,and the in vitro transcript of this fragment was ligated to circRNA circRNA-MCP by RNA ligase.The results of western blotting and cell immunofluorescence showed that circRNA-MCP could express recombinant MCP in EPC cells.Micropterus salmoides(8～12 cm long,35～55 g weight)were injected with circRNA-MCP and the results detected by qPCR showed that circRNA-MCP could enter kidney and spleen of perches.The results of ELISA showed that the specific antibody with a titer of 1:800-1600 could be detected after three-week immunization.And perches after four-week immunization vaccination were inoculated with ISKNV.The results showed that the relative immune protection rate of circRNA-MCP vaccine was 35.5%,indicating that circRNA could be designed as a new RNA vaccine against ISKNV.