Twelve reference EV71 genotypes and five recent circulating strains used for measuring cross-neutralizing antibody titers in this study.

<p>Twelve reference EV71 genotypes and five recent circulating strains used for measuring cross-neutralizing antibody titers in this study.</p

Alignment of amino acid sequences of P1 proteins of twelve reference and 5 recent strains.

<p>The P1 protein consists of VP4 (aa 1–69), VP2 (aa 70–323), VP3 (aa 324–565) and VP1 (aa 566–862). Only amino acid residues with mutation were listed. Dots indicate amino acids identical to that of A-70 strain.</p

Photographs of EV71 viral particles analyzed by transmission electron microscopy.

<p>(A) The empty particles had a defective structure with approximately 30–32 nm in diameter. (B) The full particles had a solid structure with 33–35 nm in diameter. The bar indicates 50 nm.</p

Antigenic map of twelve rabbit antisera against 11 representative genotypes and 5 recent circulating strains.

<p>The relative positions of viral strains (color points) and antisera (grey points) were adjusted such that the distances between strains and antisera in the map represent the corresponding neutralization titers. Each strain has a outer circle which points out the range of standard error. The space between grid lines is 1 unit of antigenic distance, corresponding to a 2-fold dilution of antiserum in the neutralization assay.</p

Scatter plot between ratios of homotypic and heterotypic GMTs in humans and cross-reactive antibody titers in rabbits.

<p>Based on influenza experience, ≧50% differences in GMT of cross-reactive antibody titers in humans (i.e. ≧2 in cross-reactive antibody ratios) may cause decreased vaccine efficacy <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003044#pntd.0003044-Lee4" target="_blank">[30]</a>. The horizontal and vertical dashed lines indicate cut-off of 2 in ratio of human GMTs and cut-off of 8 in ratio of cross-reactive antibody titers in rabbits. Numbers in each corner indicate true positive (upper right), false positive (lower right), false negative (upper left), and true negative (lower left).</p

Phylogenetic analysis of EV71 strains.

<p>The phylogenetic tree was constructed using nucleotide sequences of P1 genes and the reliabilities indicated at the branch nodes were evaluated using 1,000 bootstrap replications. Only bootstrap values of over 70% were shown. The prototype coxsackievirus A16 (CA16) G-10 strain was used as an out-group. Solid circle : recent strains; Solid triangle : reference strains.</p

Neutralizing antibody titers of rabbit antisera against twelve reference and five recent strains.

<p>Neutralizing antibody titers of rabbit antisera against twelve reference and five recent strains.</p

Five amino acid mutations which may be related to antigenic variation of EV71 genotype A virus are located on 3-D structure of capsid protein (PDB 3VBS) using the RasMol software (

<p><a href="http://rasmol.org/" target="_blank">http://rasmol.org/</a><b>).</b> VP1 (blue), VP2 (cyan), VP3 (green) and VP4 (yellow) proteins are presented in whole virus particle (left panel), space fill model of P1 polyprotein (upper right panel), and cartoon model of P1 polyprotein (lower right panel). Two neutralizing epitopes (VP1: 211–225; VP2: 136–151) identified using mouse monoclonal antibodies are shown in the cartoon model of P1 polyprotein.</p

EV71 virus strains used for detecting cross neutralizing antibodies in this study.

<p>EV71 virus strains used for detecting cross neutralizing antibodies in this study.</p

Cross-reactive neutralizing antibody titers in children infected with EV71 C4a and B5 genotypes.

<p>Cross-reactive neutralizing antibody titers in children infected with EV71 C4a and B5 genotypes.</p