岡本 宏明

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.

A seroepidemiological survey of four hepatitis C virus (HCV) relative antibodies was conducted among 1062 inhabitants who lived in an endemic area for non-A, non-B hepatitis in Gifu prefecture Japan, and were older than 30 years. Each seropositive rate of HCV relative antibodies was as follows: anti C100-3 antibody 15.3%, anti CP-9 antibody 36.0%, anti CP-10 antibody 33.3% and anti GOR antibody 22.3%. 482 of 1062 (45.4%) inhabitants were seropositive for anti C100-3 antibody and/or anti CP-9 antibody and/or anti CP-10 antibody, who were considered to have been infected with HCV previously. Remarkably, 91 of 918 (9.9%) inhabitants with normal serum level of ALT (&lt 36 IU/l) were seropositive for anti C100-3 antibody. © 1991, The Japan Society of Hepatology. All rights reserved.

All 92 clones of HBV-DNA propagated from the sera of 8 patients with fulminant type B hepatitis after amplification by polymerase chain reaction had a point mutation from G (guanine) to A (adenine) at position 1896 (at nucleotide 83) in the precore-region. This mutation converted codon 28 from tryptophan (TGG) to a stop codon (TAG) and inhibited the synthesis and secretion of HBe antigen. On the other hands, precore-region defects were not observed in any clones from 3 cases of acute type B hepatitis. These findings suspected that HBV infection with point mutation in the precore-region was one of the important factors to induce fulminant type B hepatitis.

Clones of hepatitis B virus (HBV) DNA were propagated from sera of two babies who developed neonatal fulminant hepatitis B, as well as from sera of their mothers who carried HBV with antibody to hepatitis B e antigen, and the precore-region sequences were determined. A point mutation from guanine to adenine, converting codon 28 for tryptophan (TGG) to a stop codon (TAG), was detected in 18 of 20 HBV DNA clones from mother and all 31 clones from baby in one family, and invariably in 55 clones from mother and three clones from baby in the other family. These results indicate that HBV mutants defective in the precore region in some carrier mothers with antibody to hepatitis B e antigen may transmit fulminant hepatitis B to their babies.

Fulminant hepatitis B developed in 8 recipients of blood units without detectable hepatitis B surface antigen on routine screening. All 124 hepatitis B virus (HBV) DNA clones propagated from their sera possessed defects in the precore region. A point mutation from guanine to adenine at nucleotide 83, converting codon 28 for tryptophan (TGG) to a stop codon (TAG), was the commonest, and it was found in all 113 clones from 7 cases. The remaining case displayed 1 clone with this point mutation and 10 clones with an insertion of 2 base pairs after nucleotide 26. Antibody to hepatitis B core antigen (anti-HBc) was detected in a high titer in 1 of 10 pilot plasma samples of blood units transfused to this case. HBV DNA clones propagated from it exhibited the same precore-region defects as those from the recipient. On the basis of these results HBV mutants, defective in the precore region, would appear to be responsible for posttransfusion fulminant hepatitis B, and the exclusion of blood units with high-titered anti-HBc would be efficacious in preventing it.