title: Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody.
abstract: The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infection; it is involved in binding to host receptors and contains specific epitopes that elicit neutralizing antibodies. However, most previous studies have not provided detailed molecular information about the functional epitopes on JEV EDIII protein. In this study, we described a monoclonal antibody (mAb 2B4) we produced and characterized by IFA, PRNT, ELISA and Western blot analyses. The results showed that mAb 2B4 was specific to JEV EDIII protein and possessed high neutralization activity against JEV in vitro. Furthermore, we found that the motif, (394)HHWH(397), was the minimal unit of the linear epitope recognized by mAb 2B4 through screening a phage-displayed random 12-mer peptide library. Using sequence alignment analysis it was found that this motif was highly conserved among JEV strains and was present in West Nile Virus (WNV). Indeed, ELISA data showed that this epitope could be recognized by both JEV-positive swine serum and WNV-positive swine serum. Notably, this linear epitope was highly hydrophilic and was located within the terminal end of a β-pleated sheet of EDIII. An analysis of the spatial conformation supported the possibility of inducing specific antibodies to this epitope. Taken together, we identified (394)HHWH(397) as an EDIII-specific linear epitope recognized by mAb 2B4, which would be beneficial for studying the pathogenic mechanism of JEV; and mAb 2B4 was also a potential diagnostic and therapeutic reagent.
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Wen-Lei
authors.initials: WL
authors.lastname: Deng
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Chi-Yu
authors.initials: CY
authors.lastname: Guan
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Xiu-Li
authors.initials: XL
authors.lastname: Feng
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China. Electronic address: zhoubin@njau.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Su
authors.affiliation: Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Pu-Yan
authors.initials: PY
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology; D004664:Encephalitis Virus, Japanese / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004672:Encephalitis, Japanese / Q000276:immunology / Q000821:virology*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D014759:Viral Envelope Proteins
keywords: EDIII protein; Epitope; Japanese encephalitis virus; Monoclonal antibody; Spatial conformation