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, 39 (12), 4370-9

Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides From HEV Genotype 4

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Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides From HEV Genotype 4

Y Wang et al. J Clin Microbiol.

Abstract

We reported previously on the complete sequence of hepatitis E virus (HEV) genotype 4, isolated from patients with sporadic cases of acute HEV infection in China. At least eight HEV genotypes have now been described worldwide, and further isolates await classification. Current immunoassays for the detection of anti-HEV antibodies are based on polypeptides from genotypes 1 and 2 only and may be inadequate for the reliable detection of other genotypes. Because genotypes 1 and 4 predominate in China, we wished to investigate the antigenic reactivities of HEV genotype 4 proteins. Four overlapping regions of open reading frame 2 (ORF2) (FB5, amino acids [aa] 1 to 130; E4, aa 67 to 308; F2-2, aa 288 to 461; E5, aa 414 to 672) and the entire ORF3 product were expressed in Escherichia coli as fusion proteins. Enzyme immunoassays based on each of the five purified polypeptides were evaluated with sera from patients with sporadic cases of acute HEV infection. Individual immunoassays derived from HEV genotype 4 detected more cases of acute hepatitis E than a commercial assay. Some serum samples, which were positive for anti-HEV immunoglobulin G only by assays based on HEV genotype 4, were positive for HEV RNA by reverse transcription-PCR. Polypeptide FB5, from the N terminus of ORF2, had the greatest immunoreactivity with sera from patients with acute hepatitis E. These data indicate that the N terminus of ORF2 may provide epitopes which are highly reactive with acute-phase sera and that assays based on genotypes 1 and 2 alone may be inadequate for the detection of HEV infection in China, where sporadic cases of HEV infection are caused predominantly by HEV genotypes 4 and 1.

Figures

FIG. 1
FIG. 1
Organization of HEV genotype 4 and locations of sequences expressed as recombinant antigens. (A) Translation strategy and ORFs of HEV genotype 4 (30). The ORF2 product has a signal sequence at the amino terminus (black box) and potential N-linked glycosylation sites (indicated by lollipop-shaped symbols). The 7.5-kb genomic RNA is shown below. (B) Magnification of the 3′ region of the genome and ORFs 2 and 3. Arrows above and below the line representing RNA show the approximate locations of the PCR primers. Shaded boxes below the ORFs indicate the regions expressed in recombinant proteins FB-5, F2-2, E4, E5, and O3.
FIG. 2
FIG. 2
SDS-PAGE of the five recombinant polypeptides. Lanes 1 to 5, purified O3, FB5, F2-2, E4, and E5 fusion polypeptides, respectively; lanes M: polypeptide molecular mass markers.
FIG. 3
FIG. 3
Dendrogram of HEV sequences generated in the present study, produced by using the PileUp program (Genetics Computer Group). All homologous genotype 2, 3, and 4 sequences present in the GenBank nucleotide database are included for comparison, along with a representative selection of genotype 1 sequences. Accession numbers for GenBank sequences are as follows: for genotype 1, India, accession no. x98292; China 1, accession no. l25547; China 2 (Xinjiang), accession no. d11092; China 3, accession no. af141652; China 4, accession no. m94177; Burma 1, accession no. m73218; Burma 2, accession no. d10330; Nepal, accession no. af051830; Pakistan, accession no. af185822; Egypt, accession no. af051352; Morocco, accession no. af065061; for genotype 2, Mexico, m74506; for genotype 3, US1, af060669; US2, af060669; for genotype 4 (all from China or Taiwan), HF-030, accession no. af134916; HF-054, accession no. af134917; TW6310E, accession no. af117279; HF-044, accession no. af134612; TW6196E, accession no. af117278; TW8-E2, accession no. af117275; TW2494E, accession no. af117276; T21, accession no. af151963; T11, accession no. af151962; T1, accession no. aj272108; LZ-105, accession no. af103940; TW5483E, accession no. af117277; TW32SW, accession no. af117280; TW74SW, accession no. af117281.

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