Development Of Inactivated Iridovirus And Rhabdovirus Bivalent Vaccine And Its Immune Response In Marble Goby,Oxyeleotris Marmoratus

Oxyeleotris marmorata,also known as Thailand Marbled sand goby,was a famous kind of cultured fishes for Eleotridae,Ophiocephalus.With the expansion of its breeding,the disease in sand goby become more and more serious,MSGIV(Marbled sleepy goby iridovirus)and OMBRV(Oxyeleotris marmoratus rhabdovirus,OMBRV)are the most serious pathogens among its aquaculture industry,in addition these two viruses often co-infected.At present,control of sand goby diseases mainly rely on antibiotics and chemical drugs.It easily leads to drug residues and screen out the drug-resistant strains,bringing serious threat to food safety and human health.However,vaccination is the most economic and effective method to prevent the viral diseases,but the sand goby vaccine has not been reported.Therefore,in this paper an inactivated bivalent vaccine was developed using MSGIV and OMBRV which originated from the sand goby.Then,the differentially expressed genes and their signal pathways induced by vaccine in sand goby spleen were analyzed by RNA-sequencing technology.At last,expression changes of the immune-related gene were determined by qRT-PCR method.These results laid a foundation to elucidate the immune response mechanism of vaccine in sand goby.The main results are as follows:1 Development and immune protection effect of inactivated iridovirus and rhabdovirus vaccine for marbled sleepy goby.CPB(Chinese perch brain cell line)cells that are sensitive to MSGIV and OMBRV were used to inoculate MSGIV and OMBRV isolated from diseased Oxyeleotris marmorata.When complete pathological changes appeared in cell,their titers were 10~7.35.35 TCID50/ml and 10~10.680.68 TCID50/ml,respectively.The viruses were inactivated by 0.2%formaldehyde.Then the inactive test was determined through cell blind passage method and the challenge test of fish.Then two kinds of inactivated virus were equally mixed,emulsified with IMS1312adjuvant according to 1:1(volume).Safety test results show that Oxyeleotris marmorata actived normally and had no clinical symptom after injection with inactivated vaccine.There was no fish death during 14 days post-vaccination,suggesting that the vaccine is safe.Immunized Oxyeleotris marmorata was challenged with virulent MSGIV and OMBRV at 28d post-vaccination.There was no clinical symptom in immunization group after challenge,and survival rate were 100%.Control group began to appear death challenged with MSGIV after 5d,and the the accumulative mortality rate was 100%.Control group began to appear death challenged with OMBRV after 1d,and the accumulative mortality rate was 100%.Death individuals showed MSGIV or OMBRV typical symptoms of infection,and result of PCR detection of MSGIV or OMBRV is positive.These result showed that relative percent survival of the vaccine for MSGIV or OMBRV were all 90%.2 Transcriptome analysis of spleen in Oxyeleotris marmorata post vaccination or post-infection.The cDNA Libraries of the spleen at 7d and 14d post vaccination and 48 h and 7 d post-infection were constructed.Total 85547 unigenes were obtained by sequencing,with 32386 annotated unigenes.Among them,total13593 differentially expressed genes(DEGs)were obtained from spleen cDNA library,including 1291 up-regulated genes and 2030 down-regulated genes at 7d post vaccination,3732 up-regulated genes and 6540 down-regulated genes at 14d post vaccination.Total 15658 and 13982 DEGs from spleen cDNA library infected by MSGIV or OMBRV,including 8073 up-regulated genes and 5595 down-regulated genes at 48 post MSGIV infection;5110 up-regulated genes and down 10371 at 7d post MSGIV infection;3797 up-regulated genes and 5846 own-regulated genes at 48post OMBRV infection;10025 up-regulated genes and 3704 own-regulated genes at7d post OMBRV infection.GO functional annotations and KEGG analysis showed that the immune-related DEGs mainly occurred in cytokin-cytokine receptor pathway,phagosome pathway,etc.Transcriptome analysis of spleen in Oxyeleotris marmorata post-immunization and post-infection provided the background informations for clarifying the mechanism of vaccine induced immune response.3.Expression analysis of immune-related gene in Oxyeleotris marmorata post vaccination or post-infection.Based on the DEGs in the transcriptome,9immune-related DEGs involved in Toll-like receptor pathway,antigen presentation and antibody production pathways were selected for expression analysis by qPCR.The expression of MHC I gene was significantly up-regulated 2.67-fold(p<0.05)and1.676-fold(p<0.05)than control at 7d and 14d post-vaccination,however there was no significant change in MHC II gene expression,indicating that the antigen was mainly presented by MHC I molecules.The expressions of TCR and IgM genes were significantly up-regulated 10.1-fold(p<0.01)and 6.8-fold(p<0.01)than control at 48h post-infection with MSGIV.And there were up-regulated 6.3-fold(p<0.05)and9.8-fold(p<0.01)than control at 48 h post-infection with infection with OMBRV,indicating that both cellular and humoral immunity all play an important role in vaccine-induced immune protection.In addition,TLR9 gene of Toll-like receptor pathway was up-regulated 7.9-fold(p<0.01)and 4.7-fold(p<0.05)than control at 48h and 7d post-infection with MSGIV.However,TLR7 was up-regulated 5.7-fold(p<0.05)and 2.6-fold(p<0.05),and TLR8 was up-regulated 10.5-fold(p<0.01)and6.8-fold(p<0.01)than control at 48h and 7d post-infection with OMBRV,suggesting that two viruses induced different innate immune responses.