Preparation And Immune Protection Effect Analyses Of An Inactivated Vaccine Against Infectious Pancreatic Necrosis

Infectious pancreatic necrosis（IPN）is a viral disease caused by infectious pancreatic necrosis virus（IPNV）,which can cause a large number deaths of salmon and trout,the mortality rate up to 100%.IPNV was imported to China from Japan in the 1980s,and it has seriously affected the development of salmon and trout cultivating industry in China.At present,there is no effective medicine for the prevention and control of IPN in China.Due to the fact that vaccine is one of the most effective methods to prevent viral diseases,and the inactivated vaccine has the advantages of safety,efficiency and simple production.In this study,an IPN inactivated vaccine was preliminary prepared.Then,the immune protection effect of inactivated vaccine was evaluated.Phylogeny analysis of IPNV strains isolated from diseased farms were performed by VP2 gene sequences.The result showed that Genogroup I and V IPNV were prevalent in China,and the distribution of Genogroup I IPNV were more widely.The IPNV strains IPNV-F1 and IPNV-W2 isolated in this study belonged to Genogroup I and IPNV-W1 isolated in this study belonged to Genogroup V.Considering that Genogroup I IPNV strains had wide distribution,which was used to prepare IPN inactivated vaccine.In order to find the optimal amplification pattern,IPNV was inoculated to Chinook salmon embryo cells（CHSE-214）to successively pass generations with 100,1000,and 10000 TCID₅₀,and the IPNV titer of each generation was determine to select optimal inoculation dose.Inoculating IPNV to cells with the optimized dose,the RNA of cells was extracted at 12-84 h post inoculation,and gene expression level of IPNV gene was quantified using the 2^-△△CT method of Real-time quantitative PCR（RT-q PCR）to select the best time for IPNV harvest.The result showed that the titer of IPNV was the highest and most stable with 100 TCID₅₀,which was selected as the optimal inoculation dose.By using this dose,gene expression of IPNV reached the highest value at 72 h post inoculation,which was selected as the optimal time for IPNV harvest.A large scale of IPNV was prepared using the optimal amplification pattern, and was inactivated byβ-propiolactone（BPL）at the final concentration of0.025-0.500%for 6-48 h,respectively.Then,the inactivation was confirmed in vitro and vivo to select the optimal inactivation conditions.The result showed that IPNV can be completely inactivated when final concentration of 0.500%BPL inactivated IPNV at room temperature for 48 h,which was selected as optimal inactivate condition.Due to no rainbow trout death when they were challenged with IPNV,the relative percent survival can’t be determined.To establish a challenge model for evaluating the immune protection efficacy of vaccine,in this study,IPNV was intraperitoneal injected into rainbow trout at a dose of 10μl per fish(1.00×10^5.00TCID₅₀ per fish),and the changes of virus titer in tissues were detected on 3,7,14,and 30 d post challenge（d.p.c）.The time point that virus titer was highest was selected for testing the protective efficacy of vaccine.The result showed that the IPNV titer reached the highest value on 3 d.p.c,which was selected as the time point for sampling after challenge.The IPN inactivated vaccine prepared by optimal inactivation conditions was intraperitoneal injected into rainbow trout（10±2 g）with 100μl per fish(1.00×10^7.50TCID₅₀ per fish),and the rainbow trout injected equal volume of PBS were as negative control.Then,on 3,7,14,30,45 and 60 d post immunization（d.p.i）,the rainbow trout were injected and challenge model was used to detect the changes of virus titer in tissues of each group.The result showed that the IPNV titer in the immune rainbow trout tissue significantly decreased during 60 d.p.i,reaching peak on30 d.p.i（p<0.05）.Gene expression of immune related factors IFN-1 and Mx-1 in spleen and head kidney from vaccinated fish was determined by the 2^-△△CT method of RT-q PCR on 3,7and 15 d.p.i,and those of CD4,CD8 and Ig M were determined on 15 and 30 d.p.i.The neutralizing antibody titer was detected on 30,45,and 60 d.p.i.The result showed that the expression of IFN-1 in the spleen and head kidney showed an upward trend,reaching peak on 15 d.p.i（p<0.05）,which was 2.46 and 2.83 folds compared with the control group.The expression of Mx-1 in the spleen increased first and then decreased,reaching peak on 7 d.p.i（p<0.05）,which was 4.30 folds compared with the control group.In head kidney,Mx-1 gene expression showed an upward trend,reaching peak on 15 d.p.i（p<0.05）,which was 41.90 folds compared with the control group.In spleen and head kidney,CD8 and Ig M genes were significantly up-regulated on 15 and 30 d.p.i,and CD4 genes were significantly up-regulated in head kidney on 15 d.p.i.The mean neutralizing antibody titers in the serum of immunized rainbow trout with a decreasing trend（p<0.05）,which were 41.2,56.4 and 36.9 on 30,45 and60 d.p.i,respectively.The above results indicated that the IPN inactivated vaccine prepared in this study has a well immune protection effect during 60 d.p.i,can stimulate the non-specific and specific immune response of immune rainbow trout,the gene expression of immune-related factors can up to 41.9 folds compared with the control group,this study laid a foundation for prevention and control of IPN in China.