Comparative analysis between HEV ORF3 protein and peptides with deduced amino acids borne by selected phages.

<p>The consensus sequence was mainly identified in the ORF3 C-terminus. Only the sequence from A93 to R114 of ORF3 is presented. “.” represents residues of deduced amino acids differing from the ORF3 C-terminus, “-”indicates no residues. The numbers at the top signify the position of the residue at the ORF3 C-terminus. The first “3” represents position 93, and the last “4” represents position 114.</p

Deduced amino acid sequences of peptides borne by selected phages.

<p>① Isoleucine (I) has similar structure and characteristics as Leucine (L)</p><p>② Isoleucine (I) has similar structure and characteristics as Valine (V). I, L and V all belong to the branched chain amino acid group. The underline indicates the continuous consensus sequence within the C-terminal region of ORF3 protein.</p><p>N/A, not applicable.</p

Efficacy of biopanning for HEV ORF3 mAbs.

<p>Efficacy of biopanning for HEV ORF3 mAbs.</p

Comparative analysis of ORF3 C-terminal amino acid sequences among the four HEV genotypes.

<p>Residues shaded in black represent those differing from genotype 1 HEV ORF3. The consensus continuous sequence "VVDLP" is presented in the red box. The number in brackets represents the GenBank accession no. of the selected virus.</p

Alternation of BM B cells populations following virus infection.

<p>Following infected with V and Ts virus, the BM cells from three mice per group per time point was prepared on day 3, 5 and 7 post-infection and the phenotypic FACS analysis of B220, IgM, IgD, CD43 expressions within BM cells was performed. (A) The numbers of B220⁺ cells within BM; (B) the percentages of BM B lineage cells within B220 gated cells populations. B220^lowIgM⁻ cells define pro-B (B220⁺CD43^high) and pre-B (B220⁺CD43^low/−) cells, B220^lowIgM⁺cells are immature B cells and B220^highIgM⁺ cells are mainly mature naive recirculating B cells. Percentages shown are calculated from total B220⁺ cells (erythrocyte-depleted). The numbers of pro- (C), pre- (D), immature B cells (E) and mature naïve B cells (F) were calculated by multiplying the percentage of each cell type by the total number of BM viable cells. Data shown in graph A, C, D, E, F represents mean ± SD for three independent experiments. Data shown in graph B are two-color dot plots from individual mouse, but are representative of three independent experiments. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V and Ts.</p

Binding analysis of selected phages to ORF3 mAb-1 using ELISA.

<p>Control and selected phage names are shown on the <i>z</i> axis, the OD₄₉₀ values of individual phages and control on the <i>y</i> axis, and logarithm value of the phage concentration on the <i>x</i> axis.</p

Role of sympathetic nervous system in the production of GCs and depletion of spleen and BM B cells.

<p>Following 6-OHDA treatment, six mice were infected i.n. with 10⁴ PFU of V (V-6-OHDA) and another six mice were infected i.n. with PBS (mcok-6-OHDA). Another two groups (6 mice/group) treated with vehicle were infected i.n. with 10⁴ PFU of V (V-alone) or PBS (Mock-vehicle), respectively. (A) GC levels in plasma of each group were measured using ELISA. After infected with V virus, spleen and BM cells were prepared on day 5 post-infection. The proportions (B) and numbers (C) of splenic B220⁺ B cells were determined. The numbers of pro-, pre-, immature B cells and mature naïve B cells from BM (D) were calculated by multiplying the percentage by number of total BM cells. Data bars represent means of 6 mice from each infection group ± SD. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V-vehicle and V-6-OHDA. Ns mean no statistical difference between both.</p

Numbers of B lineage cells in BM following treatment with RU486 and/or Leptin.

a<p>Following treatment with RU486 (R), RU486 plus leptin (RL), leptin (L), vehicle respectively, mice were infected i.n. with 10⁴ PFU of V (V-) or PBS (Mock-), respectively. The right femurs from six mice per group were collected at day 5. BM cells were prepared and stained with anti-CD45/B220, anti-CD43, anti-IgD and anti-IgM, and analyzed by flow cytometer. The numbers of different B cells populations were calculated by multiplying the percentage of each cell type by the total number of BM viable cells. Data is representative of means of six mice ± SD for three independent experiments..</p>b<p>Indicates statistically significant differences (<i>p</i><0.05) comparing to data in V-vehicle group.</p

ELISA analysis of binding of synthetic peptides to ORF3 mAb.

<p>The binding activity of each peptide is presented in individual histograms. The <i>x</i> axis represents peptide concentration and <i>y</i> axis shows the OD₄₉₀ value of each peptide at different concentrations. The color of each bar represents the ORF3 mAb serial concentration. Schematic illustration showing that only SAPPLPPVVDLPQLGL and SAPPLPPVVDLP react with the mAb,</p

Expression of three truncated ORF3 proteins and identification of the antigenic epitopes.

<p>(a) Bacteria containing three truncated ORF3 genes induced by 1 mM IPTG at 37°C for 5 h and identified via SDS-PAGE. <i>Lane M</i>, protein molecular weight marker. <i>Lanes 1</i>, <i>3</i>, <i>5</i>, uninduced bacteria containing ORF3-1, ORF3-2 and ORF3-3 truncated genes, respectively. <i>Lanes 2</i>, <i>4</i>, <i>6</i>, induced bacteria containing ORF3-1, ORF3-2 and ORF3-3 truncated genes, respectively. Arrows indicate the positions of the three recombinant proteins with a molecular weight of ~27 kDa. (b) Identification of the antigenic epitopes of ORF3 using mAb-1 via western blot. Only the ORF3-3 protein was recognized by this mAb, as indicated with the arrow. The lanes in (b) are one to one correspond to lanes in (a), but experiments were performed using two SDS gels. (c) Schematic representation of the truncated HEV ORF3 constructs and reactivity to mAbs. The names and lengths of the truncated constructs are indicated. Binding ability to ORF3 mAb-1 was determined based on western blot results. “+”, positive result and “-”, negative result. Results obtained with mAb-2 were similar to those with mAb-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133282#pone.0133282.s004" target="_blank">S4 Fig</a>), and ORF3 protein recognized by two mAbs via western blot was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133282#pone.0133282.s005" target="_blank">S5 Fig</a>.</p