Establishment And Application Of An Indirect ELISA For Detection Of Serum Antibodies Against Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(Bovine Viral Diarrhea Virus,BVDV)is a small positive-stranded RNA virus,which belongs to the genus Flaviridae.BVDV is an important pathogen associated with gastrointestinal,respiratory and reproductive diseases in cattle.BVDV can cause the decline of cattle reproductive performance,so it causes continuous economic losses to the cattle industry.The virus can cross the placenta in the early stages of pregnancy and cause persistent infection(PI)of calves.Animals with persistent infection usually transmit BVDV,more effectively than animals with transient or acute infection because they can shed virus throughout their lives and are considered to be the main hosts of BVDV.Because of the nature of virus infection,there is no way to completely cure animals infected with the virus.In recent years,domestic serological investigation shows that BVDV infection exists in almost all cattle-raising areas in China,and the infection rate is increasing year by year.In addition,the number of cattle imported from abroad,especially from Europe,Australia and other places has increased,so our country must strengthen the quarantine and prevention of BVDV.At present,virus neutralization test and imported commercial kit are mainly used in the detection of BVDV serum antibody in our country.Virus neutralization test is not suitable for large-scale detection of serum,and imported commercial kit is more expensive.For this reason,this study carried out the establishment of an indirect ELISA method for the detection of BVDV serum antibodies,in order to provide technical support for the prevention and control of BVDV in China.The most immunogenic proteins of BVDV include structural proteins Erns(E0),E2 and nonstructural protein NS3(p80),all of which can be used to detect antibodies against BVDV in bovine serum.Among them,E0 and E2 proteins are the main immunogens of BVDV,which can induce neutralizing antibodies and are often the main targets for antibody detection.P80 protein is a non-structural protein of the virus,which has strong immunogenicity.The virus can also induce antibodies against p80 in the process of replication in the body,and p80 is also the main target for BVDV antibody detection.In this study,the recombinant plasmids pET30a-p80,PET30a-E0 and PET30a-E2 were separately transformed into E.coli BL21(DE3)expression bacteria.After the transformants were cultured and induced,it was confirmed by SDS-PAGE that the three kinds of proteins E0,E2 and p80 were expressed in the form of inclusion bodies.The three purified recombinant proteins E0,E2 and p80 were used as coating antigens,and three indirect ELISA methods for detecting BVDV serum antibodies were established through square titration and optimization of reaction conditions.At the same time,the three coated recombinant proteins only reacted with BVDV positive serum,but had no cross reactions with antibodies against bovine infectious rhinotracheitis virus,bovine parainfluenza virus type 3,bovine respiratory syncytial virus,foot-and-mouth disease virus and classical swine fever virus.The sensitivity test of ELISA method with E2 or P80 as coating antigen showed that the positive serum was still positive after dilution for 1280 times,indicating that the sensitivity of the ELISA method for E2 or p80 was higher.The sensitivity test of ELISA method with E0 as coating antigen showed that the positive serum was positive after dilution for 640 times,indicating that the sensitivity of this method needed to be improved.The repeatability tests of the three ELISA methods showed that the intra-batch coefficient of variation was less than 10%,and the inter-batch coefficient of variation was also less than 10%,indicating that the three methods had good repeatability.The three methods were used to detect bovine serum and compared with virus neutralization test.The results showed that the coincidence rate between the ELISA methods for E0,E2 and p80 and virus neutralization test were 88.1%,95.1%,and 96.5%,respectively.The coincidence rate between the ELISA method for E0 and virus neutralization test was lower than that for other two ELISA methods for E2 and p80.A total of 188 bovine serum samples collected from 2 different regions in China were detected with the indirect ELISA method for p80,and the serum positivity was 86.2%.And a total of 384 bovine serum samples collected from 4 different regions in China were also detected with the indirect ELISA methods for E2 and E0,and the serum positivity for E2 and E0 were 88.02% and 87.2%,respectively.On the other hand,224 bovine serum samples collected from a cattle herd inoculated with an inactivated vaccine for BVDV were also detected with the three ELISA methods for E0,E2 and p80,respectively,and 223 bovine serum samples were positive for the three ELISA methods.Through comparision of the three indirect ELISA methds for E0,E2 and p80,especially for the lower coincidence rate between the ELISA method for E0 and virus neutralization test,the indirect ELISA methd for E2 might be more specific and suitable for the seroepidemiological investigation for BVDV,and would be used as a technical support for the prevention and control of BVDV in China.