Development Of Indirect ELISA For Detection Of Antibodies To Bovine Viral Diarrhea Virus Type ? In Pigs And Its Application

Bovine viral diarrhea virus(BVDV)may infect cattle and sheep,causing acute infectious diarrhea.Recent studies have suggested that the virus could also infect pigs,as evidenced by high seroprevalence in swine serum samples.BVDV and classical swine fever virus(CSFV)have high similarity in their genome structure and coding proteins.In addition,there are two genotypes of BVDV,BVDV type I(BVDV1)and type II(BVDV2)(or BVDV1/2 for both genotypes).Therefore,it is necessary to develop suitable immunological methods for detection of antibodies specific to particular viruses in pigs that might have immunized with attenuated live CSFV vaccine,and/or infected with field CSFV strains,or even BVDV1/2.There are reports that BVDV seroprevalence was high in swine serum samples in several regions in China.However,BVDV seroprevalence in Zhejiang remains unknown.This study was aimed:1)to develop indirect ELISA(i ELISA)assays for detection of serum antibodies specific to BVDV1/2 using prokaryotic products of E^rns and E2 proteins of BVDV1/2;and 2)to investigate BVDV1 seroprevalence of swine serum samples collected from several regions in Zhejiang by the optimized i ELISA.1.Preparation of prokaryotic products of E^rns and E2 proteins of BVDV1 and BVDV2Antigenic regions of E^rns and E2 of both BVDV1 and BVDV2 were analyzed.Molecular cloning was used to express the full-length products or their truncated regions were expressed using p ET-30a(+)as the expression vector.These recombinant expression plasmids were transferred into Escherichia coli Rosetta strain,which were then induced with IPTG(isopropyl-beta-D-thiogalactopyranoside).The prokaryotic proteins(BVDV1-E^rns-R,BVDV1-E2,BVDV2-E^rns-R and BVDV2-E2)were purified and identified by SDS-PAGE.They were then applied in subsequent development of i ELISA.2.Optimization of indirect ELISA system for detection BVDV1 antibodies in pig seraWe found that cross-reactivity to CSFV-positive sera could be significantly reduced by optimizing the E^rns and E2 antigenic region of BVDV1 or BVDV2.Then we continued to optimize BVDV1/2-i ELISA methods.However,only BVDV1-i ELISA could be possible because BVDV2 E^rns and E2 antigens were of high cross-reactivity to BVDV1-positive sera.For BVDV1-i ELISA,the E^rns and E2 antigens(5?g/m L)were coated overnight at 4^oC.The blocking agent was 0.1%casein solution.Dilutions for serum samples and HRP-conjugated secondary antibodies were 1:200 and 1:10,000,respectively.Incubation for each antibody binding was 60 min at 37^oC.The intra-assay CV for this i ELISA ranged from 2.86 to 6.86%,and the inter-assay CV,from 2%to4.13%.The average OD value plus 3SD was 0.39 for the E^rns coating and 0.49 for the E2 coating based on 31 negative serum samples.The cutoff value was set at 0.4 for E^rnsand 0.5 for E2.A commercial ELISA kit was compared with this i ELISA on 390 serum samples.When double positive findings for both E^rns and E2 were considered seropositive for BVDV1,the sensitivity and specificity(relative to commercial kit)was94.3%and 88.3%,respectively,and the Kappa value was 0.83.When E^rns and E2 were individually considered for BVDV1 seropositivity,the corresponding sensitivity was94.8%and 94.3%,the respective specificity was 87.2%and 87.8%,and the Kappa value for both antigens was 0.82.These findings suggest that either E^rns or E2 could be used alone or in combination for BVDV1-i ELISA for specific detection of BVDV1seroprevalence in swine serum samples.3.BVDV1 seroprevalence in swine serum samples collected in pig herds in different regions of Zhejiang provinceOf the 1246 pig serum samples from 2017 to 2019 collected by our lab and Zhejiang Academy of Agricultural Sciences detected by BVDV1-i ELISA,the positive rate of BVDV1-E^rns was 57.1%;E2,50.6%;and E^rns and E2 both,44.8%.Seroprevalence seemed to increase by year from 2017 to 2019,and vary among regions from 39.7%to 62%.In conclusion,the BVDV1-i ELISA established in this study can be used to investigate BVDV1 seroprevalence in swine serum samples and further developed as a possible kit after some improvement.While BVDV1 seroprevalence in Zhejiang was remarkably high,as in other provinces in China,systemic analysis and viral detection(either viral nucleic acid or virus isolation)are required in the future to determine if there are really natural BVDV1 infections in the swine herds or if these are simply the result of contamination of swine vaccines,either inactivated vaccines or live attenuated,by BVDV1.