Abstract
BackgroundThe West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported.ResultsIn this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae.ConclusionThe KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.
Highlights
The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization
The 6D3 monoclonal antibody (mAb) showed strong reactivity against WNV antigen slides by an indirect immunofluorescence assay (IFA; Figure 2, panel b)
We found that the 6D3 mAb reacted with WNV and Japanese encephalitis virus (JEV) by IFA, but not with other non-JEV serocomplex flaviviruses, such as Dengue virus 1-4 (DENV1-4), YFV and Tick-borne encephalitis virus (TBEV)
Summary
The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. West Nile virus (WNV) is a positive-sense, singlestranded RNA virus of the family Flaviviridae, genus Flavivirus. It is a member of the Japanese encephalitis virus (JEV) serocomplex, which is comprised of several medically important viruses including WNV, JEV, SaintLouis encephalitis virus (SLEV) and Murray Valley fever virus (MVEV) [1,2]. The first elucidated 3 D structure of DENV C protein dimer (residues 21-100) suggested possible mechanisms for its interactions with RNA and the viral membrane [7]
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