Establishment Of Indirect ELISA For EMCV And Preliminary Evaluation Of EMCV Inactivated Antigen

Swine encephalomyocarditis virus(EMCV)is one of the members of Cardiovirus,Picornaviridae family.EMCV genome is approximately 7.8 kb in length and is a single-stranded positive RNA,which can be translated directly as a template into a large polyprotein.Subsequently,the large polyprotein is cleaved to produce at least 13 mature proteins by the 3C protease(3Cpro).Among them,1D is the viral capsid protein,which is located on the surface of the virion.EMCV 1D interacts with the cell receptors on the cellular membrane,which is critical for the viral invasion.EMCV 1D contains many epitopes to be recognized by EMCV positive serum,Therefore,EMCV 1D is used as immunogenic protein to establish several serological detection methods.EMCV has a broad host spectrum and can infect a variety of animals,including primate.Pigs are most sensitive to EMCV.Young pigs may develop acute myocarditis with sudden death,while adult pigs often occur subclinical symptoms.Sows infected with EMCV can cause reproductive disorders.In 2005,EMCV virus was first isolated from clinical samples collected from different regions in China.Subsequently,several EMCV isolates were reported.Serological investigation results show that EMCV infection exists in large-scale farms in many regions of China,thus it is necessary to develop serological detection methods and produce vaccine to prevent and control EMCV.In this research,an indirect ELISA detection method for EMCV was established and EMCV inactivated antigen were developed and evaluated its safety and protective effect using mice as model.1.Establishment of indirect ELISA assay with EMCV 1D as immunogenic proteinEMCV 1D protein was obtained by Ni Sepharose protein purification filler using affinity chromatography in the research.The concentration of the purified EMCV 1D protein was up to 1.5 mg/mL.The 1D indirect ELISA for EMCV was established and reaction conditions were optimized.The best reaction conditions are as follows: The 96-well plates were coated with recombinant EMCV 1D with a concentration of 1 μg/mL.The serum sample was diluted with the dilution of 1:2000 and incubated for 1.5 h at 37℃.The dilution of enzyme-labeled antibody was 1:5000,and the action time was 30 minutes.Finally,the substrate acted at 37℃ for 10 minutes and the absorbance values are measured.Thus,we established an indiret ELISA detection method.2.Production of EMCV inactivated antigen and evaluation of the protection efficiencyEMCV HB10 was inactivated using BEI and mixed with adjuvant ISA 15 VG to produce inactivated antigen.Mice were immunized with different doses inactivated antigen respectively.After immunization and challenge,the 1D antibodies in the serum from the immunized mice could be detected by indirect ELISA,indicating that the inactivated antigen could stimulate the specific immune response.After challenge,mice in the control group showed obvious clinical symptoms and histopathological changes with high tissue viral load and then died.Only one mouse survived.However,the clinical and pathogenic symptoms of mice immunized with inactivated antigen with low tissue viral load were not obvious and all survived.In conclusion,the 1D indirect ELISA for EMCV were established,and the EMCV inactivated antigen could protect susceptible mice against EMCV infection,which could be used as a candidate vaccine to control EMCV.