A precore defective variant of hepatitis B virus has been indicated to cause fulminant hepatitis in various instances such as intrahospital outbreaks or mother-to-child transmission of hepatitis B virus. To learn whether similar variants are involved in interspouse transmission, we analyzed three cases of fulminant hepatitis B that developed in formerly healthy subjects whose only exposure to hepatitis B virus was contact with their longtime spouses, who were carriers of HBV and positive for antibody to HBe. The DNA clones for precore and S genes were propagated from patients and spouses and sequenced. Because of the conservation of S-gene sequences and the identity of subtypes between patient and spouse, it was suggested that patients were infected with hepatitis B virus from their spouses, not from other sources. A TGG-to-TAG mutation at the 28th codon of the precore gene of hepatitis B virus was commonly observed in all DNA clones from patients with fulminant hepatitis and from their spouses. A 29th-codon GGC-to-GAC mutation was additionally evident in DNAs from one patient-and-spouse couple. A significant rise in the circulating hepatitis B virus concentration was transiently observed in the index spouse of this case just before development of fulminant hepatitis in her husband. The increase in circulating HBV DNA was associated with a rise in abundancy of variants with mutations at both the 28th and 29th codons, compared with variants with only a 28th-codon mutation. The double mutation in hepatitis B virus DNA may either help the virus escape immune surveillance or replicate at a higher rate than before.

Base Sequence↗

PubMed1992

The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients.

We investigated amino acid heterogeneity in the variable regions of the E2/NS1 viral protein in interferon-responsive and interferon-nonresponsive patients with chronic hepatitis C virus infection. The study assessed whether any particular heterogeneity pattern(s) could be useful in predicting responsiveness to interferon treatment. The nucleic acid sequences of the hepatitis C virus genome were analyzed from six patients with chronic hepatitis treated with an interferon-beta, three of whom did not respond to the therapy and another three who showed remarkable improvement in the serum levels of liver enzymes and hepatitis C virus RNA after 6 mo. The complementary DNA clones propagated from each of the nonresponders showed significant diversity of both nucleotide and amino acid sequence, especially at the hypervariable region 1 within the putative E2/NS1 gene of the virus, suggesting that these patients were infected with a large heterogeneous pool of hepatitis C virus variants. In contrast, the responders showed little or no diversity in the sequence of the complementary DNA clones, suggesting that they were infected with one or a small population of viral genotypes containing significantly less variability in the E2/NS1 hypervariable region 1. These results suggested that a large variable population of hepatitis C virus genotypes is implicated in patients who are nonresponders to interferon treatment. In addition, a significant change in the hepatitis C virus genotype population was observed in nonresponders after interferon treatment. This may reflect a differential viral sensitivity to interferon, selective immune pressure by the host or both.

Adult↗

PubMed1992

Two distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core protein.

Four distinct genotypes of hepatitis C virus types I, II, III and IV have been identified by comparison of nucleotide sequences of isolates from different areas of the world. We examined the possibility that hepatitis C virus may have serologically definable subtypes. Enzyme-linked immunosorbent assay systems were prepared by use of two synthetic peptides deduced from the putative core protein of hepatitis C virus. The following are the two peptides that were used: (a) IPKARRPEGRTWAQPGY (subtype-1) conserved in hepatitis C virus isolates with type I and type II genotypes; and (b) IPKDRRSTGKSWGKPGY (subtype-2) conserved in type III and type IV genotypes. With the enzyme-linked immunosorbent assays, the subtype-1 antibodies were detected in 26 (68%) of 38 subjects whose hepatitis C virus RNA had been genotyped as type I or type II, whereas subtype-2 antibodies were not detected. Inversely, the subtype-2 antibodies were detected in 10 (56%) of 18 subjects with hepatitis C virus RNA genotypes III or IV, whereas subtype-1 antibodies were detected in none of them. These results suggest that hepatitis C virus has two serologically distinguishable core antigen subtypes, corresponding to either genotype I/II or genotype III/IV. Subtyping of HCV by serological methods would contribute to tracking transmission routes of the virus, especially in cases where serum samples were not stored under conditions to preserve RNA or in infected hosts who have cleared the virus and therefore have only antibodies remaining to identify the infection.

Amino Acid Sequence↗

PubMed1992

Immune response to GOR, a marker for non-A, non-B hepatitis and its correlation with hepatitis C virus infection.

Recently, identification and molecular cloning of a host cellular gene designated GOR from chimpanzees experimentally infected with non-A, non-B hepatitis (NANBH) agent was reported. It was further demonstrated that there is a close association between the immune response to an antigenic peptide of GOR (GOR2) and NANBH. In order to define the specificity of the immune response, in the present study we have identified an additional epitope in the GOR gene sequence, upstream from GOR2, and studied its correlation with the immune response to hepatitis C virus (HCV) in NANBH patients. An enzyme-linked immunoassay (EIA) was developed which utilizes synthetic peptides designated spGOR346 and spGOR2 as the serological target for the detection of anti-GOR antibodies in patient serum samples from various hepatic and non-hepatic disease categories. GOR peptides identified 80-90% of the NANBH samples that were positive for HCV C100-3 and about 70% of the NANBH samples that were positive by Abbott prototype second-generation HCV antibody assay. Among a normal donor population(s), only 2-3% of the samples were positive for antibodies to GOR sequences, whereas from the patient categories unrelated to viral hepatitis as well as various nonhepatic diseases, the immune response to both GOR peptides was closely associated with the presence of antibodies to HCV. The data indicate that antibodies to GOR is a marker associated with NANBH.

Amino Acid Sequence↗

PubMed1992

p26 protein and 33-nm particle associated with nucleocapsid of hepatitis C virus recovered from the circulation of infected hosts.

Hepatitis C virus (HCV) has not yet been cultured or visualized. We attempted to recover HCV-associated particles from plasma of infected humans to assess the natural properties of the virus. Starting with 720 ml of donor plasma containing high titer of HCV core antigen ELISA activities, we identified HCV core antigen activity and viral RNA enriched in a potassium bromide density gradient fraction with a density of 1.115 g/ml. Icosahedron-shaped particles with an average diameter of 33 nm were liberated by treatment of the fraction with the detergent Tween 80. These particles were selectively visualized with an electron microscope using a grid coated with a murine monoclonal antibody directed to HCV core peptide and were also observed in aggregated forms with an immune electron microscope (IEM) with use of the anti-core antibody. An ultracentrifugation pellet of the above fraction was treated with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2ME) and run in SDS-PAGE. A protein that bound antibodies directed to the predicted core protein of HCV was found at a molecular size estimated as about 26,000 Da, significantly greater than the 191 amino acid residues predicted from the presumed core gene of HCV. It is possible that translation initiation and/or the COOH-terminal cleavage site for HCV core protein in vivo may differ from estimates derived from the amino acid sequence of the polyprotein precursor. The nucleocapsid could also be chemically altered in the infected cell, resulting in a gel mobility different from the native protein.

Enzyme-Linked Immunosorbent Assay↗

PubMed1992

Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes.

Variable genomic sequences have been reported for RNA cloned from hepatitis C virus (HCV)-infected humans and chimpanzees. We found that four distinct genotypes of HCV could be differentially identified by PCR using type-specific primers. Full-length sequences have so far been reported for three of the four HCV genotypes, and we report herewith the sequence of the fourth type obtained from a Japanese blood donor. The entire nucleotide sequence of the HCV isolate (HC-J8) comprised 9481 bases plus a 3'-terminal poly(U) stretch of variable length. Like all previous isolates, the RNA contained a single, long open reading frame for a polyprotein of 3033 amino acids. HC-J8 differed from previously reported HCV isolates by 23.1-33.1% in nucleotide sequence and 15.9-28.8% in amino acid sequence. Based on genomic sequence homologies, a proposed phylogenetic tree of HCV, with a fourth branch represented by HC-J8, allowed a classification of all HCV isolates whose complete or partial sequences are now known. This classification suggests that all or most HCV genome sequences will fall into one of the proposed four types. The classification may be helpful in designing vaccine studies and for serological investigations of possible group- and type-specific antibodies.

Amino Acid Sequence↗

PubMed1992

Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability.

Extensive variability in genomic sequence, especially at "hypervariable regions" within the NS1/E2 region of the long open reading frame, has been reported for RNA cloned from hepatitis C virus (HCV)-infected humans and chimpanzees. However, genetic changes of HCV occurring during the course of chronic infections in humans and animals have been evaluated only for partial sequences of the HCV genome. We compared two full-length cDNA sequences of HCV obtained from a chimpanzee that was experimentally infected with the HC-J4 strain of HCV: one during the early acute phase and another during a chronic phase 8.2 years afterward. Both isolates had 9412 nucleotides plus the 3' poly(U) tail with varying length organized as follows: 5'UTR (1-341); C (342-914); E (915-1490); NS1/E2 (1491-2528); NS2 (2529-3359); NS3 (3360-5186); NS4 (5187-6380); NS5 (6381-9371); and 3'UTR (9372-9412). We found that 111 (1.18%) of the 9412 nucleotides differed between the two isolates and estimated the mutation rate as approximately 1.44 x 10(-3) base substitutions per site per year. Changes in amino acid coding were associated with 42 mutations, 8 of which were clustered at 5' end of NS1/E2 coding region, so-called "HVR-1." We analyzed the HVR-1 and HVR-2 sequences during the course of infection and found that homologous populations were present at the beginning of infection, and sequence heterogeneity within the region had developed 3.5 years later. Two regions of the HCV genome were characterized by a high degree of conservation of nucleotide sequence: 5'UTR and the 3' half of the NS4 region. The possible secondary structure of the 5'UTR suggests a region for internal ribosomal entry. The 3' half of the NS4 region may also have some specific function which depends upon a strict conservation of nucleotide sequence.

Amino Acid Sequence↗

PubMed1992

Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts.

An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.

Amino Acid Sequence↗

PubMed1992

Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction.

The genomic RNA of hepatitis C virus (HCV) in the plasma of volunteer blood donors was detected by using the polymerase chain reaction in a fraction of density 1.08 g/ml from sucrose density gradient equilibrium centrifugation. When the fraction was treated with the detergent NP40 and recentrifuged in sucrose, the HCV RNA banded at 1.25 g/ml. Assuming that NP40 removed a lipid-rich surface coat from HCV, the 1.08 g/ml and 1.25 g/ml HCV RNA may correspond to intact HCV virions and nucleocapsids, respectively. The extraordinarily low density of the virion is unusual in comparison to the density of classified viruses.

Base Sequence↗

PubMed1992

Mutations within the S gene of hepatitis B virus transmitted from mothers to babies immunized with hepatitis B immune globulin and vaccine.

A variant of hepatitis B virus (HBV) having a specific mutation within the S gene has been found to infect vaccinees. To know whether similar variants were involved in Japan, we analyzed two cases of maternal transmission of HBV in infants immunized with hepatitis B immune globulin and hepatitis B vaccine. DNA clones of HBV S genes were propagated from patients and family members and sequenced. In one family, the DNA clones from the baby patient had a Gly-to-Arg mutation at the 145th codon of the S gene, whereas those from her mother had no such mutations. In the other family, all the DNA clones obtained from the two infected children had the 145th codon intact, but they had a missense mutation at the 126th codon of the S gene, causing an amino acid substitution of Asn for Thr or Ile. This same mutation was observed in 12 of 17 clones of DNA obtained from their mother. In comparison with the wild type HBV-derived hepatitis B surface antigen, the two types of S gene mutations, either at the 145th or the 126th codon, were associated with a significant decrease in the antigenicity of some determinants on the hepatitis B surface antigen, measured by MAb. Amino acid substitution at these sites, therefore, would have induced the escape from conventional vaccines that were S gene products of wild type HBV and also from hepatitis B immune globulin, whose main components were probably also antibodies against the S gene products expressed by wild type HBV.

Adult↗

PubMed1992

Hepatitis C virus infection in medical personnel after needlestick accident.

Hepatitis C virus infections in medical personnel after needlestick accidents have been documented generally by detection of seroconversion to a hepatitis C virus nonstructural region antigen, c100-3 (a marker of infection). We tested for hepatitis C virus core-derived antibodies and genomic RNA in addition to c100-3 antibody in 159 cases of needlestick exposure that did not involve patients positive for HBsAg. Of these we found 68 cases with index patients positive for both hepatitis C virus RNA and antibodies and members negative for antibodies to HCV core or c100-3 before the needlestick accidents. Seven of these medical personnel became infected with hepatitis C virus after the accidents. Their hepatitis was generally subclinical or self-limited and transient, except for one patient in whom liver enzyme elevation persisted along with the antibodies. In our study, the risk of hepatitis C virus transmission from a single needlestick accident with hepatitis C virus RNA-positive blood was 10%, considerably higher than the 4% estimated in a previous study. We found that donor blood with antibody to an hepatitis C virus core-derived peptide with enzyme-linked immunosorbent assay optical densities greater than 2.0 carried a significant risk of transmitting hepatitis C virus to needlestick victims. No hepatitis C virus seroconversions occurred in medical personnel exposed to hepatitis C virus antibody-negative or hepatitis C virus RNA-negative blood; however, one such exposure resulted in a very mild non-A, non-B, non-C hepatitis.

Health Personnel↗

PubMed1991

An autoantibody cross-reactive to hepatitis C virus core and a host nuclear antigen.

GOR, an epitope borne by the amino acid sequence, GRRGQKAKSNPNRPL, is recognized by anti-GOR antibodies specifically found in patients with non-A, non-B hepatitis (NANBH). The epitope is not coded for by the hepatitis C virus (HCV), the presumed causative agent for NANBH, but by a single copy gene of the host. Anti-GOR antibodies, distinct from anti-HCV (c100-3) antibodies, were revealed to have dual specificities; they target both the presumed core gene product of HCV and a host component. This cross recognition is probably derived from homologous regions between the GOR epitope and a viral epitope on the core protein in HCV. It is therefore suggested that anti-GOR is an autoantibody induced by HCV infection. This may explain the autoimmune disease like aspect of NANBH pathogenesis.

Amino Acid Sequence↗

PubMed1991

[A non-A non-B hepatitis specific epitope GOR and its antibodies].

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Amino Acid Sequence↗