岡本 宏明

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.

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.

Three hepatitis B virus carriers who were HB(e)Ag negative and having normal liver function developed fulminant hepatitis with evidence of HBV replication following intensive chemotherapy for non-Hodgkin's lymphoma. Each was continuously negative for HB(e)Ag. Analysis of the precore region of HBV isolated from each demonstrated that the HBV of each had a point mutation in the precore region that inhibited the synthesis and the release of hepatitis B(e) antigen. This observation suggests that all HB carriers receiving either immunosuppressive or cytotoxic therapy should be monitored closely even if standard assays suggest that viral replication is not present. Sudden enhanced replication of a HBV mutant as a result of such therapy can be a cause of either very severe hepatitis or occasionally fulminant hepatitis.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p <0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p <0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p <0.001). Sera with HBsAgl/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p <0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.

The putative core gene of hepatitis C virus (HCV) was incorporated into a plasmid vector (pCC5-J4), and expressed in Escherichia coli. The product of 180 amino acids (p20c) was purified by gel electrophoresis in the presence of sodium dodecyl sulfate, and used in enzyme-linked immunosorbent assay for antibodies against the putative core protein of HCV (anti-p20c). Anti-p20c was detected in 13 (1.5%) of 873 apparently healthy blood donors. It was detected in 205 (86.5%) of 237 patients with acute or chronic non-A, non-B (NANB) hepatic disease, significantly more frequently (p < 0.01) than antibodies against the C100-3 protein encoded by nonstructural regions of HCV (anti-C100-3) that was found in 178 (75.1%). Anti-p20c developed in the circulation of a patient with acute NANB hepatitis much earlier than anti-C100-3. HCV RNA was detected by polymerase chain reaction in serum samples from blood donors positive for anti-p20c in high titers, one of which was negative for anti-C100-3. These results indicated that anti-p20c would be useful in complementing anti-C100-3 for the diagnosis of NANB hepatitis and further decreasing the incidence of posttransfusion NANB hepatitis.

Based on variation in nucleotide sequence within restricted regions in the putative C (core) gene of hepatitis C virus (HCV), four groups of HCV have been postulated in a panel of 44 HCV isolates. They were provisionally designated types I, II, III and IV. A method for typing HCV was developed, depending on the amplification of a C gene sequence by polymerase chain reaction using a universal primer (sense) and a mixture of four type-specific primers (antisense). HCV types were determined by the size of the products specific to each of them. Type II was found in HCV samples from 131 (82%) of 159 blood donors, more often than in those from 48 (60%) of 80 patients with non-A, non-B (NANB) liver disease in Japan (P < 0.01). In 11 haemophiliacs who had received imported coagulation factor concentrates, type I was found in five, as against type II in four. Double infection with two different HCV types was found in two patients with chronic NANB liver disease (types I and II; II and III) and two haemophiliacs (types I and II; I and III). HCV types were identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle exposure.

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.

Immunoassays were developed to detect antibodies against oligopeptides deduced from the putative core gene of hepatitis C virus, and their performances were compared with that of the commercial immunoassay for antibodies against the product of nonstructural regions of hepatitis C virus (anti-C100-3). A 19-mer oligopeptide (CP10) and a 36-mer oligopeptide (CP9) were chemically synthesized, which represented hydrophilic regions of the product of the hepatitis C virus core gene. They were used to capture corresponding antibodies, anti-CP10 and anti-CP9, by enzyme-linked immunosorbent assay in sera from patients with acute or chronic non-A, non-B liver disease and in blood donations. At the onset of acute non-A, non-B hepatitis, anti-CP10 was detected in 15 of 20 patients (75%), and anti-CP9 was detected in 14 patients (70%). This was more frequent than anti-C100-3, which was found in only 9 patients (45%). In 186 patients with chronic non-A, non-B liver disease, anti-CP9, anti-CP10 or both were detected in 170 patients (91%). This was more frequent than anti-C100-3, which was found in 138 Patients (74%). Blood with anti-CP10 as the single serological marker for hepatitis C virus infection transmitted non-A, non-B hepatitis by needlestick exposure. In sera from 558 apparently healthy blood donors, anti-CP10 was detected in 55 donors (9.9%), anti-CP9 was detected in 26 donors (4.7%) and anti-C100-3 was detected in 7 donors (1.3%). Among sera Positive by at least one test, 14 were found to contain hepatitis C virus RNA; 7 of them were negative for anti-C100-3 but positive for anti-CP10 and/or anti-CP9 in high titers. These results indicate that antibodies against antigenic determinants of the hepatitis C virus core would complement anti-C100-3 for the diagnosis of non-A, non-B liver disease and contribute toward further decreasing the incidence of posttransfusion non-A, non-B hepatitis.

Hepatitis B virus (HBV) DNA was tested for in 294 blood units which had antibody against hepatitis B core antigen (anti-HBc) as the isolated serological marker of HBV infection. After amplification by polymerase chain reaction, HBV DNA was detected in 12 (6.9%) of 175 units that were positive for anti-HBc with hemagglutination inhibition titers greater-than-or-equal-to 2(6), significantly more often than in none of 119 units with titers less-than-or-equal-to 2(5) (p < 0.01). These results indicate that the exclusion of blood units with isolated high-titer anti-HBc would be effective for further decreasing the risk of posttransfusion hepatitis B.

The complete nucleotide sequence of a hepatitis C virus derived from plasma of a human carrier in Japan was determined. The cDNA of the isolate (HC-J6) contained 9481 nucleotides and an additional T stretch of 30 to 108 nucleotides at the 3' end, and had one large open reading frame coding for a polyprotein of 3033 amino acids. It differed by 31.8 to 32.1% in the nucleotide sequence and by 27.4 to 27.7% in the amino acid sequence from an American isolate and two Japanese isolates previously reported. Among these four isolates, the 5' non-coding region of 329 to 341 nucleotides was well conserved (> 93% identity), whereas the 3' non-coding region of 39 to 45 nucleotides (T stretches not included) was more variable (> 30% identity). An excellent degree of conservation of the 5' non-coding region would reflect its pivotal role in replication, and primers deduced from this region could be applied for the sensitive and specific detection of viral RNA by polymerase chain reaction. Due to a high degree of similarity in the amino acid sequence of the putative core protein (> 90%), antigen probes deduced from it would be suitable for the serological diagnosis of HCV infection. Low sequence similarity in the putative envelope protein (> 53% identity), however, would have to be taken into account in considering the immunoprophylaxis of HCV infection.

The S-gene sequences of hepatitis B virus (HBV) from 22 carriers in several islands of Indonesia were amplified by polymerase chain reaction, and XbaI-SpeI fragments corresponding to nucleotides 93-529 (437 base pairs) in the S gene were sequenced. The 22 sequences, along with the 5 reported sequences from Indonesia, were compared with each other, and with the corresponding sequences of 20 clones from other countries including China, France, Great Britain, Japan, Kenya, Papua New Guinea, Philippines, USA and USSR. When the 27 HBV DNA clones of various subtypes from Indonesia were classified by the homology in the nucleotide sequence into the five genotypes, twelve belonged to genotype B (subtype adw 7 and ayw 5), 13 to genotype C (adw 1, adr 10, ayr 1 and ar 1), and 2 to genotype D (ayw); none belonged to genotype A or E. Different subtypes of clones in the same genotype indicated that point mutations inducing d-to-y or w-to-r phenotypic changes would be common among Indonesian carriers. Comparison of the translation products of XbaI-SpeI fragments, now available for 47 HBV DNA clones of different genotypes (A 4; B 14; C 21; D 7; E 1), identified several amino acids characteristic to or influenced by the five genotypes as well as those highly conserved by clones of different genotypes.

We have assayed HCV RNA levels, anti-CP-9 and anti-C100-3 in serial serum samples from 12 patients with chronic hepatitis C who were treated with short term (4-6 weeks) interfelon. In six patients, serum ALT levels fell to normal and remained in normal range after termination of treatment (effective group). The ALT levels did not improve in other six patients (non-effective group).<BR>Serum HCV RNA decreased markedly in all patients belonged to both groups at the end of treatment. In effective group. HCV RNA in serum sustained (-) or (+) after stopping interferon, but HCV RNA in serum returned gradually or rapidly to pre-treatment levels ( - ) in non-effective group. These observations suggest that interferon seems to suppress HCV RNA in serum and interferon treatment may improve natural course of chronic hepatitis C.