Insight into SNPs and epitopes of E protein of newly emerged genotype-I isolates of JEV from Midnapur, West Bengal, India

Abstract

Background: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese Encephalitis (JE) and Acute Encephalitis Syndrome (AES) in humans. Genotype-I (as co-circulating cases with Genotype-III) was isolated in 2010 (JEV28, JEV21) and then in 2011 (JEV45) from Midnapur district, West Bengal (WB) for the first time from clinical patients who were previously been vaccinated with live attenuated SA14-14-2 strain. We apply bioinformatics and immunoinformatics on sequence and structure of E protein for analysis of crucial substitutions that might cause the genotypic transition, affecting protein-function and altering specificity of epitopes.

Results: Although frequency of substitutions in E glycoprotein of JEV28, JEV21 and JEV45 isolates vary, its homologous patterns remain exactly similar as earlier Japan isolate (Ishikawa). Sequence and 3D model-structure based analyses of E protein show that only four of all substitutions are critical for genotype-I specific effect of which N103K is common among all isolates indicating its role in the transition of genotype-III to genotype-I. Predicted B-cell and T-cell epitopes are seen to harbor these critical substitutions that affect overall conformational stability of the protein. These epitopes were subjected to conservation analyses using a large set of the protein from Asian continent.

Conclusions: The study identifies crucial substitutions that contribute to the emergence of genotype-I. Predicted epitopes harboring these substitutions may alter specificity which might be the reason of reported failure of vaccine. Conservation analysis of these epitopes would be useful for design of genotype-I specific vaccine.

Keywords: B-cell & T-cell epitopes; Docking; Genotype I; Genotype III; Homology model; Japanese encephalitis virus; Midnapur; PEP-FOLD; SNP Energetics.

Fig. 1

Comparison of net substitutions for each domains and total of E protein among JEV28 (yellow), JEV45 (red) and Ishikawa isolates (cyan). JEV21 is not included in the plot as it is identical to JEV45

Fig. 2

Residual plot of average physicochemical properties for GI isolates (Ishikawa, JEV28 and JEV45) in reference to SA14-14-2. Analyses were performed using PHYSICO [17] and PHYSICO2 [18] programs with an input of E proteins of SA14-14-2, Ishikawa, JEV28 and JEV45 in FASTA format. Percentile difference for each property was computed against the reference data and plotted using SYSTAT Sigma Plot v11.0

Fig. 3

Backbone Cα-traces of homology model (a for Ishikawa, b for JEV45 and c for JEV28) of the ecto domain of PM0080325, PM0080324 and PM0080323 respectively. Domain I (red), domain II (yellow) and domain III (blue) are shown in different colors. Fusion loop (purple) and substitutions with respect to the template (SA14-14-2) are highlighted in each of the model based on their occurrence (see Table 1). Disulfide bonds (green) are also highlighted in each of the model based on their occurrence and only labeled in case of JEV45. Comparison of ANOLEA-profiles [61] of PM0080324 (green trace) with template (red trace) (D1), VERIFY3D (D2) analysis [31] and Ramachandran plot (D3) of main chain dihedral angles (core region is outlined in deep blue and allowed region in red) for residues (glycine as green circle and non-glycine as pink points) of the model along with PROCHECK [29] analysis (D4) are presented for model validation

Fig. 4

B cell specific conformational antigenic determinants (AI) and linear epitopes (AII). (AI, AII) In each case domains of E protein are presented by different colors (i.e. domain I red, domain II silver and domain III green). (B) Representative Epitope pairs (BI for G388K and BII for W396R) for comparison of main chain topology of wild-type (blue) and mutant-type (red). The wild-type (yellow) and mutant (cyan) type residues are shown in stick formats. Each of this peptide structure was generated using PEP-FOLD [43] followed by normalization and fixation of charge and potential. RMSD of mutant structure was computed in reference to its wild-type. F-L: Fusion loop; RGD-L: RGD loop

Fig. 5

Docking and structure based short listing of antigenic epitopes using human class I MHC (HLA-A i.e. 2X4O.pdb in a) and class II MHC (HLA-DRB1 i.e. 1DLH.pdb in b) crystal structures. Typical sequence and structure of docked peptide (blue color A & B) with white one as mutated residue. PBG indicates peptide binding groove which was mapped using NACCESS procedure [44] (see Materials and Methods for details). Two views per MHC class are shown for better visualization of binding pocket and binding groove. Chains of both classes of MHCs are shown by conventional notations