Detection of hepatitis G virus (HGV) RNA: clinical characteristics of acute HGV infection.

The role of hepatitis G virus (HGV) infection in acute non-A-E hepatitis was investigated in adults with viral hepatitis. HGV RNA was present in 1 of 28 patients with non-A-E hepatitis but 9 of 22 with hepatitis C (P < .003). HGV RNA-positive patients (HGV-infected and HGV-hepatitis C virus [HCV]-coinfected) developed light-to-moderate jaundice. Clinical and biochemical features of HGV-positive and HCV-positive patients and patients with non-A, non-G hepatitis were similar. Three patients with HGV-HCV coinfection, tested within 18 months after disease onset, have remained HGV RNA-positive but have become HCV RNA-negative. Only 1 non-A-E hepatitis patient was confirmed as being infected with HGV alone, suggesting that HGV is not the main etiologic agent of non-A-E hepatitis. Although HGV RNA was significantly associated with hepatitis C, patients with mixed HCV-HGV infections did not demonstrate a more severe course of disease than did patients with HCV infection.

IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein.

To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immunodominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77-100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1-16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30-40 days after onset of jaundice and decreased to 1:1,778 3-4 months after the onset of jaundice. For patients tested 6-8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1-40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3-4 and 6-12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases.

Prevalence of hepatitis C virus and distribution of its genotypes in Northern Eurasia.

We tested hepatitis C virus (HCV) antibody in 4216 sera collected from healthy people living in European part of Russia (including Northern, North-Western, Central, Central-Blacksoil, Volga-Vyatka, Volga, and North-Caucasian regions), non-European part of Russia (the Urals, East-Siberia, and the Far-East regions) and Mongolia. Prevalence of HCV antibody varied significantly by regions, ranging from 0.7% in Central region of European part of Russia to 10.7% in Mongolia. Genotyping of HCV (into 1a, 1b, 2a, 2b, and 3a) was performed on 469 sera from blood donors and patients (in Russia, Moldova, Turkmenistan, and Mongolia) who were positive for both HCV antibody and RNA. Genotype 1b was the most dominant genotype irrespective of regions (68.9%), with the highest rate in Moldova (96%). HCV unclassifiable into genotypes 1a-to-3a was found in 28 (6.0%) samples: particularly 4 of 10 samples from Lipetzk were untypable. Overall, HCV genotypes in European part of Russia were more similar to those in European countries, while those in Eastern part of Russia more similar to China or Japan. Genotype distribution was not associated with the clinical expression of HCV disease: acute hepatitis, chronic hepatitis or liver cirrhosis.

Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides.

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.

Serologic identification of hepatitis E virus infections in epidemic and endemic settings.

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.

Hepatitis C virus in the etiology of chronic hepatitis and liver cirrhosis: possibility of mixed viral infections due to parenteral transmission.

Sera obtained from 381 patients with chronic liver disease from four cities within the USSR were studied for HBV, HDV, and HCV markers of infection. Anti-HCV activity was detected in 41.2% of non-A, non-B cases. The etiological distribution of chronic hepatitis in Moscow and Dushanbe was similar with an approximate 20% prevalence for HBV, HDV, and HCV infections, whereas in Yakutsk 40% of cases were caused by HDV infections. The etiology of disease remained unrecognized in approximately 40% of patients with chronic liver disease in Moscow and Dushanbe and in 15% in Yakutsk. Anti-HCV activity was detected in 18.8% of patients with chronic HBV infections and in 8.3% of patients with chronic HDV infections. Anti-HCV activity was detected in 41% of patients without markers of HBV or HDV infections. The reasons for the observed differences in HCV prevalence among patients chronically infected with HDV are discussed.