Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections

Abstract

Background: Differential diagnose of Japanese encephalitis virus (JEV) infection from other flavivirus especially West Nile virus (WNV) and Dengue virus (DV) infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV.

Results: To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120) was detected by enzyme-linked immunosorbent assay (ELISA). By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118). This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera.

Conclusion: Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

Figure 1

Short peptide designing, expression and purification. (A) Schematic diagram of the relative location of the truncated prM/M protein fragments and overlapping short peptides, M1-M20, spanning the prM/M protein. The numbers in parentheses indicate the amino acids located at the beginning or the end of each fragment. M1 to M20 are a set of partially overlapping short peptides covering the whole prM/M protein of JEV. (B) Expression and purification of recombinant peptide fusion proteins. For each peptide a fusion expression recombinant plasmid was constructed and transformed into host cell E. coli BL21 (DE3). After the cells were induced with IPTG, the supernatants of the sonicates were purified by affinity chromatography. The purified proteins were analyzed by 12% SDS-PAGE and stained with Coomassie brilliant blue. M stands for the protein molecular standards as labeled on the left.

Figure 2

Identification of the antigenic determinants on the prM/M protein with JEV-positive swine sera. (A) ELISA analysis of the short fusion peptides. Microtiter plates were coated with purified recombinant fusion protein samples (2 μg/100 μl per well). The plate was blocked with skim milk and pooled JEV-positive swine sera (dilution, 1:200) was added, following which HRP-coupled goat anti-pig IgG secondary antibody was added. Only peptide M14 showed strong reactivity with the JEV-positive swine sera. (B) Western blot analysis confirmed the result of ELISA. JEV-positive swine sera could react with GST-M14 but not with other peptide fusion protein and GST.

Figure 3

The epitope did not react with WNV-positive and DENV-positive sera. The synthesized epitope peptide was used as coating antigen in indirect ELISA. The reactivity of the epitope with the rabbit sera against JEV and that of the rabbit sera against DENV were assessed. Rabbit sera against JEV and normal rabbit sera were used as the positive and negative controls, respectively.

Figure 4

Immunofluorescence analysis of epitope-specific mice monoclonal antibody 2F7. Immunofluorescence assay of the harvested and fixed BHK21 cells infected with JEV with epitope-specific MAb 2F7 as the primary antibody and a FITC-conjugated goat anti-mouse IgG as the secondary antibody revealed that MAb recognized JEV infected BHK21 cells (A) but not uninfected cells (B).

Figure 5

Finer mapping the epitope M14-13. Peptide M14 was truncated from the carboxy and amino terminal. After short protein fragments were fusion expressed with GST, they were analyzed by ELIZA using MAb 2F7. When 2 or more amino acid residues were removed from the carboxy terminal and 4 or more amino acid residues were removed form the amino terminal, the reactivity between MAb 2F7 and peptide fusion protein decreased greatly. This result shows that peptide M14-13 (KEAWLDSTKAT) is the minimal requirement for MAb 2F7 to recognize the linearized epitope.

Figure 6

Sequence alignment of the epitope M14-13 with different JEV strains. Full-length sequences of 23 different JEV strains were selected from the GenBank. GenBank accession numbers are listed in the parentheses. Epitope M14-13 was highly conserved across these JEV strains.

Figure 7

Specificity analysis of the epitope. (A) Sequence alignment between the identified linear epitope and the homologous regions of WNV and DENV. The virus strains listed in this figure were selected as representative strains. Abbreviations of each virus and GenBank accession numbers are listed in the parentheses. (B, C) The epitope homology peptides of WNV were expressed and analyzed by ELISA using MAb 2F7. The homology peptide of WNV strain NY99 did not react with MAb 2F7, while the homology peptide of WNV strain 956 was weakly reactive with MAb 2F7 (B). JEV-positive swine sera also did not reactive with the homology peptide of WNV (C).