Development And Application Of An Antigen Capture ELISA For Detection Of Bovine Ephemeral Fever Virus Antigen

Bovine ephemeral fever?BEF?,which is caused by BEFV,is an acute febrile disease mainly in cattle,dairy cattle and water buffalo.The disease is clinically characterized by high fever,anorexia,muscle stiffness,ocular and nasal discharge,lameness,abortion and reduced milk production in dairy cattle.The disease distributes widely in more than 40 countries in Africa,Oceania,Asia and Middle East.In China,the disease has been reported in yellow cattle,buffalo,cow and yak in Taiwan Province and26 provinces in mainland.A few of assays including virus isolation,RT-PCR and real-time RT-PCR for the nucleic acid,micro-neutralization test and antibody detecting ELISA have been developed for the laboratory diagnosis of BEF.However,no assay or commercial kit is available for detecting BEFV antigen,which makes that it is difficult to perform virus screening from field samples.In the present study,the target antigenic G protein of BEFV was selected and its coding region was harvested by RT-PCR amplification with specific primers,using RNA extracted from BEFV JT02L isolate.The obtained amplicon was cloned into pGEM-Teasy vector for sequencing.The open reading frame?ORF?of G gene from JT02L isolate is 1872bp in length and encodes 623 amino acids,sharing identical antigenic sites of G1,G2,G3 and G4 with LYC11,Henan2012 and Shandong2011.Subsequently,a fragment of 1842bp that encoding all the 4 antigenic sites was sub-cloned into pPRO_EXHTb expression vector,followed by DNA sequencing and transformation into BL21?DE3?for protein expression.A target protein with a molecular weight of about 70 Ku was harvested by inducing the recombinant bacteria with IPTG and purifying with Ni-NTA system.Rabbit polyclonal antibody with high titer and good reactivity was produced by repeated immunization of New Zealand White rabbits with the purified protein.An antigen capture ELISA was developed by utilizing the polyclonal antibody coated to ELISA plate as capturing antibody and the monoclonal antibody against G1 site as detecting antibody.The assay was optimized with capturing antibody,detecting antibody and goat anti-mouse IgG-HRP in the respective dilution of 1/1000,1/1200 and 1/5000.Cross-reactivity with vesicular stomatitis virus?VSV?,inactivated Rabies virus antigen,and bovine viral diarrhea virus?BVDV?was not observed.A total of 224 whole blood or anti-coagulated blood samples from white yaks were collected from seven towns in Tibetan Autonomous County of Tianzhu,Wuwei,Gansu Province and subjected to detection with the antigen capture ELISA assay,with combination of indirect ELISA for antibody against BEFV and real-time RT-PCR.The antibody against BEFV was detected from white yaks from all the seven towns while the viral antigen was not detected.A total antibody prevalence of 45.09%?101/224?was observed,demonstrating high discrepancy among that in individual town?31.03-66.67%?.BEFV RNA was detected in 37.5%?84/224?of the samples by real-time RT-PCR.A 420bp fragment of G gene 3'terminal was amplified and sequenced for phylogenetic analysis,which showed that the BEFV infection in white yaks is closely related to JB76H that was isolate from China mainland in 1976 and belonged to subtype I.The utility of the developed antigen capture ELISA in identifying BEFV during virus isolation was further evaluated.The JT02L BEFV,which was passaged in cattle,was used to experimentally infect two bulls by intravenous inoculation.Anti-coagulated blood was collected during febrile period when the bulls displayed rectal temperature from 39.0 to 42.0°C,and used for lymphocyte separation,followed by inoculation into MDBK cells,blind passages and detection by antigen capture ELISA.The BEFV antigen was detected in the 3rd passaged cell culture.The result was confirmed by detecting the full-genome of BEFV in the cell culture.The full-genome of JT02L was shown to have 14941 nt in length and share similar genome structure with that available from Gen Bank.However,distinct features including longer coding regions of?3 and?,and a 38 AT-rich sequence in the intergenic region of?-?,which may have implications for viral virulence,were observed in JT02L strain.These results indicated that the antigen capture assay had proper utility for BEFV antigen detection,which may be helpful foundation for further BEFV screen and identification.