Identification Of Antigenic Epitopes Of Duck Tembusu Virus Envelope Protein And Development Of Epitope-ELISA Diagnostic Method

Duck tembusu virus infection is a contagious disease caused by the duck tembusu virus?DTMUV?.DTMUV disease mainly causes a severe drop in egg production of the laying ducks,a sudden decline of feed uptake and hyperemia,hemorrhage and degeneration in ovarian follicle.The disease has caused serious economic loss to duck industry in China.As an emerging infectious disease,the biological characteristics of the virus have been widely studied,but epitopes on E protein have never been documented.In this study,a 12 mer phage peptide library was used to screen B-cell epitopes on E protein with monoclonal antibodies?mAb?against DTMUV E protein.After binding,elution and amplification process,positive clones were selected by ELISA and then for DNA sequencing.The amino acid sequences of the E protein mimotopes were determined according to the positive clones sequence results.The oligonucleotide sequences for epitopes were synthesized and expressed with GST protein in E.coli.Western-blot analysis identified three minimal liner antigenic epitopes ²²¹LNLPW²²⁶,³⁷⁴EVEPPFG³⁸⁰and ⁸⁷YAEYI⁹¹.To assess the level of conservation of the epitopes among the DTMUVs,we aligned the E protein amino acid sequences.This sequence alignment revealed that the amino acids in the LNLPW,EVEPPFG and YAEYI epitope regions were identical among the DTMUV strains.The3D structure of E protein analysis indicated that LNLPW and YAEYI epitopes were located on domain II and EVEPPFG was located on domain III.To determine whether the Lx LPW,ExEPPFG and YAEYI sequences are also conserved among the E proteins of other flaviviruses,we aligned the E protein amino acid sequences of four strains of DENV,two strains of WNV,JEV and ZIKV.The result revealed that LxLPW and ExEPPFG epitopes were highly conservative in flavivirus E protein,but YAEYI not.Dot blotting assay showed that DENV-,JEV-,WNV-,ZIKV-positive sera reacted with ExEPPFG and LxLPW epitope peptides,suggesting that ExEPPFG and LxLPW were cross-reactive epitopes for West Nile virus?WNV?,Dengue virus?DENV?,Japanese encephalitis virus?JEV?and Zika virus?ZIKV?,and YAEYI was DTMUV type-specific.The diagnosis method of Epitope-ELISA was established by using type-specific YAEYI epitope and used to test duck serum samples which also detected by the Neutralization Test as the standard method.According to results,the specificity of this test was 100%.The sensitivity of this epitope-based peptide serologic test for DTMUV infection was 96%.Therefore,Epitope-ELISA was a specific method for detecting antibodies against DTMUV,which would be useful for the clinical diagnosis and evaluating the antibody level of DTMUV.