Establishment And Preliminary Application Of Indirect ELISAs Based On E0 Antigens For Detection Of Antibodies Against Classical Swine Fever Virus

Classical swine fever is an acute,febrile,and highly contagious infectious disease caused by the classical swine fever virus(CSFV),which causes severe economic losses to swine industry in the world.CSFV is a single-stranded RNA virus with an envelope.Together with bovine viral diarrhea virus(BVDV)and border disease virus(BDV),it belongs to the genus of Flavivirus within the family of Flaviviridae.CSFV has only one serotypeand can be divided into 3 genotypes.Each genotype is further divided to 3 to 4 subgenotypes.At present,although hog cholera lapinized virus is effective in preventing and controlling the spread of the disease,CSFV is still an important epidemic that significantly affects the development of the pig industry in China.Monitoring antibody levels of CSFV in swine herds,grasping the infection status of swine herds,and evaluating the efficacy of vaccine immunization are of great significance for the prevention and control of CSFV.The indirect ELISA is effective to detect antibodies against CSFV,however,it could not distinguish whether the antibodies in pigs were induced by field virus infection or vaccine immunization.In recent years,with the gradual promotion and application of E2 subunit vaccines in China,traditional antibody detection methods were not able to effectively differentiate the subunit E2 vaccine antibodies from the antibodies elicited by field CSFV strain infection.To monitor the CSF vaccine antibody levels in pig herds and effectively distinguish subunit E2 vaccine antibodies from antibodies by field strains,an indirect ELISA method was established for detection of CSF serum antibodies based on E0 protein.The primers for E0 gene were designed based on the nucleotide sequence of the CSFV in GenBank,and used to amplify E0 gene fragment.After the fragment was cloned into pGEX-4T-1 and pET-28 a vectors,the prokaryotic expression plasmids pGEX-4T-1-E0 and pET-28a-E0 were generated and identified,respectively.The expression of recombinant E0 proteinwas induced by IPTG.The purified GST-E0 and His-E0 recombinant proteins were used as antigens for the establishment of the indirect ELISA to detect the antibodies against CSFV.The concentrations of the coating antigens for GST-E0 and His-E0 were optimized by square matrix titration to be 50 ng/well and 100 ng/well,respectively,with a serum dilution in 160 ×.By statistical analysis of 80 CSFV-negative serum samples,the thresholds were defined for indirect ELISA based on GST-E0 and His-E0 as 0.167 and 0.176,respectively.Results from the specificity,sensitivity and reproducibility tests showed that the established indirect ELISA method was specific,sensitive and reproducible.Detection of the 80 serum samples using GST-E0,His-E0 ELISA and IDEXX detection kit showed the coincidence rates of the indirect ELISA method based on GST-E0 and His-E0 recombinant proteins were 78.41% and 84.09%,respectively,with that of IDEXX detection kit.The indirect ELISA method of His-E0 was more consistent than the indirect ELISA method based on GST-E0.To validate the above results,immunofluorescent assay was employed to detect the negative and positive serum samples using CSFV infected cells as antigen.The results showed that the coincidence rates of IFA assay with His-E0 ELISA and IDEXX detection kit are93.18% and 90.91%,respectively,indicating the consistency and advantage of His-E0 ELISA method over the IDEXX detection kit.The results of this study provided technical means for the surveillance of classical swine fever antibodies to identify the presence of wild virus infection in pigs immunized with E2 subunit vaccines.