Preparation And Immune Effect Evaluation Of Inactivated Vaccine Against Infectious Pancreatitis Virus

Infectious Pancreatic necrosis（IPN）is a viral disease caused by the Infectious Pancreatic necrosis virus（IPNV）,It has brought serious economic losses to the global trout aquaculture industry.IPNV was introduced into China from Japan in the 1980s,which seriously affected the development of salmon and trout aquaculture in China.Vaccine is one of the most effective methods to prevent viral diseases.Inactivated vaccine has the advantages of safety,high efficiency and simple preparation.In this study,inactivated IPN vaccine was preliminarily prepared,and its immune protection effect was analyzed.Chinook salmon embryo cells（CHSE-214）and Rainbow trout gonad cells（CHSE-214）RTG-2)is a sensitive cell line for the detection of infectious pancreatic necrosis virus（IPNV）recommended by national testing standards.Screening sensitive cell lines is the prerequisite for vaccine preparation.In order to compare the sensitivity of two cell lines to IPNV,24STRAINS of IPNV were selected in this study and treated with 1000 times of 50%Tissue culture Infective dose.TCID50)were inoculated into CHSE-214 and RTG-2 cells,respectively,and the sensitivity of the two cells to IPNV was compared by cytopathic conditions.Then IPNV was inoculated with 10TCID₅₀ and 100TCID₅₀ into the screened most sensitive cell lines,and the cytopathic conditions were observed under microscope every day,and virus cultures were harvested.Real-time quantitative PCR（REAL-TIME quantitative PCR）was used.RT-qPCR and virus titer assay were used to analyze the proliferation of IPNV on sensitive cells to determine the sensitive cell lines.IPNV was cultured and harvested on a large scale,and inactivated at 25℃with different concentrations of formaldehyde for 12 h,24h,36 h and 48 h,respectively.The optimal inactivation conditions were determined by detecting the inactivation effects at cell and animal levels.IPN inactivated vaccine was prepared on a large scale.The inactivated vaccine was intraperitoneally injected into immunized rainbow trout（Oncorhynchus mykiss）at a dose of 100μL/tail(1.0×10^6.0TCID₅₀/tail)with PBS as negative control group.The change of viral load after immunization was detected.The expression of immune-related factors and serum neutralizing antibody titer were used to analyze the protective effect of the vaccine.1:The results of sensitivity analysis showed that all 24 strains could Proliferate on CHSE-214 cells and only 8 strains of IPNV could Proliferate on RTG-2 cells at 7 d after inoculation,indicating that CHSE-214 cells were more sensitive to IPNV than RTG-2 cells.RT-qPCR and TCID50 methods were used to further analyze the Proliferation of IPNV.It was found that with the increase of inoculation time,the amount of virus strains increased first and then decreased,and reached the maximum at 3 days after inoculation except 1 day after inoculation.The amount of virus in 10TCID₅₀ group was significantly higher than that in100TCID₅₀ group（P<0.05）The amount of virus at other time points was proportional to the amount of inoculation.The results of this study suggest that CHSE-214 cells are more suitable for in vitro isolation and culture of IPNV than RTG-2 cells,and it is suitable to collect virus cultures for virus detection when typical cytopathic changes occur after inoculation.2:IPNV was cultured on a large scale with the best virus inoculation scheme,and inactivated at room tem Perature（25℃）with the final concentration of 0.025%,0.075%,0.25%,2.5%and 25%formaldehyde for 12,24,36 and 48 h,respectively.The inactivation effect was detected at cell and animal levels.The optimal inactivation conditions were determined as follows:100μL/tail dose(1.0×10^6.0TCID₅₀/tail)was used to inoculate rainbow trout（10±2 g）with intraperitoneal injection,and the same amount of phosphate buffer treatment group was set as negative control group.The results showed that IPNV could be completely inactivated only with 0.25%formaldehyde inactivated for 12 h at room temperature.The inactivated virus was transferred to CHSE-214 cells blind for the third generation,and the cell state was observed.The optimal inactivated dose was the one without any changes in the cells.In accordance with the verification conditions at the cell level,the verification at the animal level was carried out,and the abnormal feeding behavior of rainbow trout,clinical symptoms of IPN and adverse reactions caused by vaccination were observed every day.Therefore,the o Ptimal inactivation dose of formaldehyde was determined to be0.25%,and the Optimal inactivation time was 12 h.3:In order to develop inactivated vaccine for the Prevention of infectious pancreatic necrosis and evaluate its immune Protection effect,different concentrations of formaldehyde were used to inactivate at 24℃,and the best inactivation conditions were determined after safety test verification.Inactivated IPN vaccine was prepared on a large scale.The IPNV virus stock was injected intraperitoneally into immunized rainbow trout（Oncorhynchus mykiss）at a dose of 100μL/tail(1.0×10^6.0TCID₅₀/tail).PBS was used as negative control group.The expression of immune-related factors and serum neutralizing antibody titer were used to analyze the Protective effect of the vaccine.The results show that:In 1 and 2 months after immunization,rainbow trout were inoculated with IPNV virus stock solution at a dose of 100μl/tail(1.0×10^6.0TCID₅₀/tail).Three days after immunization,rainbow trout were inoculated with IPNV virus stock solution and sam Ples were collected to detect the changes of viral load in rainbow trout tissues.The virus titer decreased significantly after 60 days of immunization（P<0.05）.The results showed that IPNV inactivated solution could effectively reduce the viral load in the immune trout within 60 days after immunization,and had a continuous immune protection effect on rainbow trout,among which the antiviral ability was the strongest at 30 days after immunization.RT-qPCR results of immune-related genes showed that IFN-αand MX-1 genes were significantly upregulated in spleen of rainbow trout in immunized group compared with negative control group on day 3,7 and 10 after immunization（P<0.05）,and the expression multiple was the highest on day 10,when the maximum expression multiple was 9 times and 6times,respectively.IFN-αgene was significantly up-regulated in head kidney of immunized rainbow trout on day 7,10,14 after immunization（P<0.05）,the expression multiple was the highest on the 10th day（6 times）,and MX-1 gene was significantly up-regulated on the 3rd,7th and 10th days after immunization（P<0.05）.These results indicate that IPN formaldehyde inactivated vaccine can induce nonspecific immune response on the third day after immunization.IgM gene was significantly up-regulated in spleen and head kidney of rainbow trout in immunized group on day 14,21 and 28（P<0.05）,and the expression multiple was the highest（4 times）on day 21.In s Pleen of immunized group,gat A-3 and Granzyme A genes were significantly up-regulated at 3,7,10,14 and 21 d after immunization（P<0.05）,the gene expression multiple of GATA-3 gene was the highest on the 7th day after immunization（3x）,and the gene expression multi Ple of Granzyme A gene was the highest on the 10th day after immunization（9x）.In immunized group,gat A-3 and Granzyme A genes were significantly up-regulated at 3,7,10,14 and 21 days after immunization（P<0.05）,and the highest expression factor was 14 on day 10.The serum IPNV neutralizing antibody titers of rainbow trout at the first and second months after immunization were 98.9 and 98.2 respectively,showing a decreasing trend（P<0.05）.The results showed that IPN-formaldehyde inactivated vaccine could induce specific and non-specific immune responses of rainbow trout,and had significant immune protection effect on rainbow trout.This study screened IPNV sensitive cell lines and successfully prepared a laboratory product of IPN inactivated vaccine with good protective effect,in order to lay a foundation for the immune prevention and control of IPN disease in China.