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Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive for detecting anti-HEV activity in the sera of patients with acute hepatitis E, convalescents, and among healthy individuals.

Recombinant chimeric protein C2 containing the N-terminal region of trpE (37 kilodaltons [kDa]) and the C-terminal half (46.8 kDa) of the polypeptide encoded by ORF2 of the hepatitis E virus (HEV) genome was used for the construction of a Western blot diagnostic test for IgG and IgM antibodies to the virus (anti-HEV). (The C2 protein and the trpE protein devoid of C2 activity and used as a control for non-specific reactions were purified by recovery from sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and used for preparation of strips). Specificity of the test was proven with sera obtained from patients with acute hepatitis non-A, non-B, non-C (NANBNC) from outbreaks in different geographic regions of the world. IgG antibodies reactive to the recombinant C2 protein were detected in 93% of patients with acute hepatitis NANBNC and remained detectable in 89-100% of these patients 1-24 months after onset of jaundice. IgM antibodies were detected in 73% of patients within 26 days after onset of jaundice, in 50% 1-4 months after onset, in 6% 6-7 months after onset, and in no patients by 8 months after onset. When this test was used to identify sporadic hepatitis E cases in different regions of the world, such cases were found almost exclusively in areas where outbreaks of the disease had occurred and rarely in any other regions.