Construction And Immunogenicity Analysis Of Recombinant SVA Of Chimeric FMDV Epitopes

Senecavirus virus disease was first introduced into China in 2015.It is a swine viral infectious disease caused by Seneca virus A?SVA?.It is characterized by nose kiss and coronal vesicle lesions of hoof,and it is difficult to distinguish from Foot-and-mouth disease in clinic.At present,the disease has changed from sporadic distribution in some areas to a severe epidemic disease,and seriously affected the pig industry as there are no commercially available vaccine.The inactivated vaccine SVA CH/FJ/2017 strain developed in our laboratory has been proved to be safe and effective.As to effectively prevent and control FMD and Senecavirus diseases in pig production,a recombinant inactivated vaccine used SVA CH/FJ/2017 as vector and together with chimeric O-type FMDV epitopes was developed to achieve the purpose of"one vaccine to prevent two diseases".At the same time,it also provides theoretical basis and technical support for the further research of Picornaviridae chimeric viruses and chimeric vaccine.The main contents and results are as follows:1.Construction and Identification of Recombinant SVA for Chimeric FMDV EpitopeUsing the SVA reverse genetic operating system established in our laboratory,using the full-length c DNA infectious clone of SVA CH/FJ/2017 strain as the skeleton,the B cell epitopes of O-type FMDV structural protein VP1?135-160 amino acids and 200-amino acids213 amino acids?and the T cell epitopes of non-structural protein 3A?21-40 amino acids?were repeatedly tandem and inserted into the SVA genome with albumin signal peptide?Human albumin signal peptide,HAS?to construct a recombinant plasmid containing FMDV epitopes.The recombinant plasmid was transfected into IBRS-2 cells to rescue the recombinant virus for 25 generations.The recombinant virus was identified by genetic stability,IFA and Western blot,and the biological characteristics of the recombinant virus were analyzed by TCID₅₀,plaque and one-step growth curve.The results showed that the recombinant plasmids p SVA-HAS-OB containing B cell epitopes and p SVA-HAS-OB-3AT,containing B cell epitopes and T cell epitopes were successfully constructed and the recombinant virus was saved and named r SVA-HAS-OB and r SVA-HAS-OB-3AT.After25 passages of r SVA-HAS-OB and r SVA-HAS-OB-3AT in IBRS-2 cells,the inserted antigenic epitope sequence of type O FMDV stably existed,and the plaque phenotype and growth curve were similar to those of parental virus?r SVA CH/FJ/2017?,and the virus content was 10^8.0TCID₅₀/100mL and 10^8.13TCID₅₀/100mL.2.Pathogenicity of Recombinant virus to PigsIn the pathogenicity test of recombinant virus and parent virus to fattening pigs,the virus was challenged by neck muscle at the dose of 6m L/head.The clinical symptoms were observed and the neutralizing antibody of SVA in serum was determined.The results showed that at the same challenge dose,1/3 pigs in r SVA-HAS-OB and r SVA-HAS-OB-3AT groups had clinical symptoms,while 3 pigs in r SVA CH/FJ/2017 group had no disease.In r SVA-HAS-OB,r SVA-HAS-OB-3AT and r SVA CH/FJ/2017 groups,SVA neutralizing antibody in infected serum began to turn positive on the 5th day after infection,and the titer of SVA neutralizing antibody increased to more than 1024 on the 9th and 13th day after infection.3.Analysis of immunogenicity of recombinant virusThe inactivated vaccines were prepared from recombinant virus and parent virus respectively,and the fattening pigs were inoculated with 5m L/head dose and neck muscle route,and the second immunization was carried out at an interval of 35 days after the first immunization.The serum SVA neutralization antibody titer was detected by virus neutralization test and the serum FMDV-VP1 antibody was detected by indirect ELISA method to evaluate the immunogenicity.The results showed that the neutralizing antibody titers of SVA were all 1024 in r SVA-HAS-OB,r SVA-HAS-OB-3AT and r SVA CH/FJ/2017groups on the 14th day after the first immunization,and FMDV-VP1 specific antibodies were detected on the 28th day after the second immunization in r SVA-HAS-OB and r SVA-HAS-OB-3AT groups.To sum up,using the SVA reverse genetic operating system established in our laboratory,the recombinant SVA viruses?r SVA-HAS-OB and r SVA-HAS-OB-3AT?which can express O-type FMDV epitopes were successfully constructed and rescued.The obtained recombinant virus has similar proliferation characteristics to the parent virus r SVA CH/FJ/2017.The results of pathogenicity test showed that the pathogenicity of recombinant virus to pigs was slightly stronger than that of parent virus.In addition,r SVA-HAS-OB and r SVA-HAS-OB-3AT can induce pigs to produce high levels of SVA neutralizing antibodies and FMDV-VP1 antibodies.