Preparation And Immune Protective Efficacy Analysis Of An Inactivated Vaccine Against Infectious Hematopoietic Necrosis

Infectious hematopoietic necrosis（IHN）,caused by infectious hematopoietic necrosis virus（IHNV）,is an acute viral disease that causes high mortality in salmon and trout,especially in fry,in which mortality could be as high as 100%.IHN is highly pathogenic,highly lethal,widely distributed and rapidly spread,which causes great economic losses to the global salmon and trout industry every year.Vaccination is the best strategy for control of IHN.But to date,only Canada has a commercial IHN vaccine.Inactivated vaccines have become one of the first choices for vaccine products controlling viral diseases due to their high antigen content,stable quality,few heterologous materials,high safety,and low production cost,and they occupy the largest proportion of the approved vaccines.In order to effectively prevent the outbreak of IHN in China,we prepared an inactivated vaccine by using a Chinese IHNV isolate,and its immune protective efficacy was comprehensively analyzed in this study.In order to enrich the detection methods of IPNV,the gene sequence（711 bp）encoding VP3 protein of IPNV was amplified by one-step RT-PCR using genomic RNA of Genogroup 1 IPNV strain Ch Rtm213 as template.The VP3 protein was expressed in E.coli Rosetta.The purified VP3 protein was used as antigen protein to prepare mouse antiserum.Indirect immunofluorescence antibody test（IFAT）was used to identify the ability of antiserum to recognize the current IPNV isolates in China.The SDS-PAGE results showed that the purified VP3 protein was a single band with a size of 45 k Da,which was consistent with the theoretical size of the VP3 protein.IFAT results showed that the prepared mouse antiserum could specifically recognize Genogroup 1 and Genogroup 5 IPNV isolates those were isolated from different regions in China.Moreover,the VP3 mouse antiserum did not cross react with infectious hematopoietic necrosis virus（IHNV）and viral hemorrhagic septicemia（VHSV）,showing high specificity with IPNV.These results indicated that the VP3 protein expressed in the prokaryotic expression system had promising immunogenicity,and the prepared IPNV VP3 mouse antiserum could specifically recognize IPNV isolates from different regions in China,which laid a foundation for the immunological detection of IPNV in China.The objective of the present study was to prepare an inactivated vaccine against infectious hematopoietic necrosis（IHN）and evaluate its protective immunity in rainbow trout（Oncorhynchus mykiss）.In this study,infectious hematopoietic necrosis virus（IHNV）were successively cultured on Epithelioma papulosum cyprinid（EPC）cells with different multiplicity of infection（MOI）.The optimal proliferation patterns of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combining with the virus harvest time.IHNV prepared with the optimal proliferation pattern was inactivated byβ-propanolactone（BPL）at different final concentrations at 24℃,and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition.Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected to rainbow trout（10±2 g）with different doses,and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival（RPS）after challenge,expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination.It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC.We chose MOI of 0.0001 as the best inoculation dose on EPC cells,and the virus was harvested on 3 days post inoculation at 15℃,and IHNV titer could be steady at10^7.50TCID50/0.1 mL.The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24℃for 24 hours with the final concentration of 3.0 mM BPL.10μL per fish was chose as the optimal immunization dose,and more rainbow trout were immunized.The RPS was 91.37%,84.28%,84.15%and 47.5%on 7,21,45 and 60 days post immunization（d.p.i）,respectively,and significant difference was observed on RPS between 60 d.p.i and other time points（P<0.0.5）.Compared with the negative group,the expression levels of Mx-1 and IFN-γwere significantly up-regulated in spleen and head-kidney on 7,15 and 30 d.p.i（P<0.0.5）,and reached the maximum on 7 d.p.i（5 folds）.The expressions of CD4 and Ig M gene were significantly up-regulated in spleen and head-kidney on 15 d.p.i（P<0.0.5）.In the detection of neutralizing antibody titer,the average neutralizing antibody titer in rainbow trout serum was 67.25,43.40 and 29.78 on 30,45 and 60 d.p.i respectively,with a decreasing trend and significant differences within each group（P<0.05）.The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in rainbow trout,and could provide significant immunoprotection,which will lay foundation for the prevention and control of IHN in China.In summary,a proliferation pattern of IHNV on EPC cells,with small inoculation dose,short culture time,and stable virus titer,was developed in this study.An efficient laboratory product of IHN inactivated vaccine was successfully prepared,which will lay foundation for the prevention and control of IHN in China.