Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein

Hepatitis E virus (HEV) strains are classified into 4 genotypes by nucleotide sequencing. Genotypes 3 and 4 infect humans and animals via HEV-contaminated food or water. HEV RNA was detected by PCR and antibodies were detected by ELISA. Since human studies showed that HEV IgG antibodies in sera can persist for extended periods, diagnosis of HEV infection in swine or humans is mainly based on serological detection using commercial ELISA kits. However, there is no supplemental method to verify ELISA results. Hence, we developed a novel method used for mutual correction of these common processes. Here, a modified stable HepG2 cell line was transfected with pcDNA3.1-ORF3 to express the swine HEV ORF3 protein. Based on this cell line, a novel immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against HEV. The results show that this method has good specificity, sensitivity and repeatability. When used to investigate 141 porcine serum samples, the IPMA had a coincidence rate of 92.2% with a commercial ELISA kit. The established IPMA described herein is valuable as a supplemental method to ELISA and can differentiate infections by HEV and other viruses

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

ELISA binding analysis of mutant S⁹⁷APPLPPVVDLP¹⁰⁸ peptides to ORF3 mAb.

<p><b>A</b>ntigen names are shown on the <i>x</i> axis and OD₄₉₀ value of each peptide on the <i>y</i> axis. Schematic illustration showing that the P100A mutant maintains similar binding activity to mAb as wild-type SAPPLPPVVDLP. “**” represents significant differences (<i>P</i><0.01).</p

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

Compliance between commercial ELISA kit and ORF2 peptide-based ELISA.

<p>Compliance between commercial ELISA kit and ORF2 peptide-based ELISA.</p

Detection of BHK cells transfected with pcDNA3.1-ORF3 plasmid via indirect immunofluorescence assay using mAb-1.

<p>BHK cells transfected with pcDNA3.1 empty plasmid were used as control. Nuclei were stained with DAPI. 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.s003" target="_blank">S3 Fig</a>).</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