Analysis Of Epitopes Of Avian Hepatitis E Virus ORF3Protein From China

In2001, avian hepatitis E virus (HEV) was isolated and characterized from biles ofchickens with hepatitis-splenomegaly (HS) syndrome by Haqshenas et al. In2010, theChinese strain of avian HEV was first isolated by Zhao Qin and the full length sequences ofthis isolate shared approximately77.3%-95.5%nucleotide sequence identites with those ofother avian HEV strains. Avian HEV infection causes hepatitis-splenomegaly syndrome,hepatonecrosis, hepatic hemorrhage, regressive ovaries and upgrade mortality for chickens.Avian HEV infection is endemic in chicken flocks and has spread to North America, Europe,and China. The average percentage rate of anti-avain HEV antibody from heathy chickens inChina was about30%.To analyze the antigenicity of the ORF3protein of avian HEV, the complete and fivetruncated ORF3proteins were expressed and three monoclonal antibodies (Mabs)(6E9,3F3and2C9) against ORF3protein was generated. Their immunoglobulin subclasses were IgG1,IgG2b, IgG1, respectively. Mabs3F3and2C9recognized the same epitopes and6E9recognized overlapping epitopes with3F3and2C9, as measured by competitive enzymelinked immune response (ELISA). A main epitope localized at amino acids (aa)75-88wasidentified by indirect ELISA and Western blot using these Mabs and truncated proteins. Todetermin additional epitopes, clinical chicken sera were analyzed by indirect and comparativeELISA and Western blot using the truncated proteins. About44%of clinical chicken serawere inhibited by Mab2C9. These results showed that aa75-88of avian HEV ORF3was theimmunodominant epitope and aa1-27and aa55-74were potential minor epitopes.To analyze the antigencity of ORF3protein, eukaryotic expression protein of avianhepatitis E virus (HEV) ORF3of China isolate (CaHEV) was obtained. ORF3gene wascloned into pFastBac-HT. The recombinant plasmid was transformed into DH10Bac andtransfected into sf9insect cells to express the recombinant ORF3protein. The recombinantORF3protein was identified by SDS-PAGE, Western bolt and IFA method. The purifiedORF3protein was used to immunize BALB/c mice to produce polyclonal antibodies and thento analyze the epitopes in ORF3combing with3truncated ORF3proteins. The results showedthat ORF3protein was expressed with the highest expression level at4days post inoculation.The titer of anti-ORF3polyclonal antibodies reached104with the ELISA method. Thedominant epitopes of ORF3were located between amino acids74and88in C-terminal region.Recombinant ORF3protein from avian HEV China isolate was expressed successfully and the dominant epitopes were located between amino acids74and88in C-terminal region.ORF3shares partial sequence with ORF2, encoding a small phosphoprotein withunknown function. Because of the antigen of ORF3and ORF2, they were used for theantibodies against HEV detection. Several epitopes domains had been maped on avain HEVORF2and the relationship with HEV had been analyzed, but we know little about ORF3. Weanalyzed the antigenicity of the ORF3protein of avian HEV. Three Mabs specific for theORF3protein were successful preparation which lays the foundation for further studyregarding the construction and the biological characteristics of avian HEV ORF3. Theseresults paved the way for future study of the structure and function of ORF3protein.