Isolation Of Bovine Ephemeral Virus And Establishment Of Indirect ELISA Method

Bovine ephemeral fever(BEF)is an acute infectious disease caused by bovine ephemeral fever virus(BEFV)infection,which mainly causes sudden high fever(above40°C),rapid breathing,stiffness,and limp.And other symptoms.The incidence rate is high,the infection is strong,and the mortality rate is low.However,due to the disease,the cattle will die and the production performance will be reduced,which often causes a very serious economic loss.The bovine ephemeral fever is mainly controlled by vaccines.There is no specific medicine to treat,and the technology of vaccines needs to be strengthened.For the serological test of BEFV,the micro-serum neutralization test was still the international gold standard for detecting BEFV and the other methods were not ideal for large-scale detection.But this method required difficulty opteration and due to the time consuming,it's not suitable for field testing.Therefore,it is necessary to strengthen research on new technologies,developed new vaccines,reduced immunization time and immunization dose,increased immune protection time,and developed stable and reliable detection technology for comprehensive prevention and control of bovine ephemeral fever.In view of the current status of BEF,this study aims to collect isolated strains in the wild,and established a convenient,specific and sensitive G₁gene indirect ELISA assay.In this study,we first collected bovine anticoagulant from a herd of suspected infected cattle in Guangdong,identified by PCR sequencing and carried out genetic evolution analysis and homology analysis of BEFV G gene sequence,found that the current BEFV in China's mainland belong to one big branch and the strain is closely related to the JT02 strain in Gansu,and has a long relationship with the domestic vaccine strain JB76H.The strain is closely related to the strains popular in Taiwan,Turkey and other regions in China,and has a close relationship with the Australian strains.Compared with the recently popular strains in China,the strain has the highest homology with the recently popular JT02 strain in Gansu,with a homology of 97.6%,and the homology with the G gene sequence of the current world strain is 89.9%.?97.9%between.By passage in the brain of the suckling mouse and identificated by PCR,it was finally determined that the mouse adapted strain was obtained.Then,the rat brain suspension was infected with BHK-21 cells,and the strain was infected with the cells and then sectioned.The bullet-like virus particles were observed under scanning electron microscope.The virus titer of the cell line was 10^-4.75TCID₅₀/100?L,indicating cell-adapted strains were obtained.The successful isolation and stable passage of the cytotoxic strains in this study can complement the BEFV strains in China and lay a good foundation for further research on new vaccines.In this study,the prokaryotic expression plasmid p MAL-c5x-G₁was constructed using p MAL-c5x prokaryotic expression vector.After induction for 6 h at 27°C and a final concentration of 0.2 m M IPTG,the soluble G₁gene was successfully expressed.Recombinant proteins of MBP tags and His tags.The recombinant protein was purified by nickel column method using GE's His packing.After purification,the recombinant protein was successfully expressed by Western Blot,and the tagged protein and G₁protein were successfully expressed,indicating that the protein has good antigenicity,which laid a good foundation for further establishment of indirect ELISA.In this study,the antigen coating concentration and serum dilution were optimized by matrix titration.The value of OD₄₅₀nm of positive serum was about 1.0,the value of P/N was the largest and the background value was low.The optimal conditions were as follows:the coating antigen G₁concentration was 25 ng/100?L,the serum dilution was 1:25,the enzyme standard secondary antibody dilution was 1:10000,and the blocking solution was5%skim milk powder.The inter-assay coefficient of variation of the batch of serum was between 6.18%and 11.9%,and the intra-assay coefficient of variation was between 3.07%and 9.06%,indicating that the method has good stability.The bovine serum verified by micro-neutralization test was tested by this method.The OD₄₅₀nm result was measured and the ROC curve was analyzed by Med Calc software.The results showed that the critical value of the method was 0.347,the sensitivity was 96%,and the specificity was 92%.This indicates that the method has good sensitivity and specificity.The ELISA method established in this study has better sensitivity,specificity and stability,and the amount of antigen coated is small,which greatly reduces the cost.The method was convenient to operate,biosafety,suitable for industrialization and use,and was expected to be a standard method for detecting BEFV serum neutralizing antibodies.