岡本 宏明

An outbreak of hepatitis B virus infection occurred in a nursing facility; it involved 31 patients with sequelae of cerebral vascular accidents (15 men and 16 women; mean age, 77.4 +/- 9.3 yr). HBsAg disappeared within 6 mo in 9 patients and persisted during an observation period of more than 6 mo in 13; the remaining 9 patients were lost to follow-up while they carried HBsAg. Thus 13 of 22 patients followed (59%) became HBsAg carriers. We amplified a part of the S gene (436 nucleotides) with polymerase chain reaction on hepatitis B virus DNA from 12 randomly selected patients. The sequences of nine patients were the same as that of a nursing assistant who was an HBsAg carrier and suspected as the source of infection; it differed by only 1 or 2 (<0.5%) nucleotides from those of the remaining three patients. Between the group of nine patients with transient HBV infection and the 13 patients with persistent HBV infection, we found no differences in age or sex or in parameters of nutrition or immunocompetence. These results indicate a high incidence of HBV carrier state in the elderly.

Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that antigenic determinants of both HBV and HCV cores were accessible on them. Proteolytic digestion deprived chimeric core particles of the antigenicity for the HCV core without affecting that of the HBV core, confirming the surface exposure of HCV core determinants. The density of HCV core determinants on chimeric core particles increased as copies of fused HCV core protein were increased. Hybrid core particles with multiple HCV core determinants would be instrumental as an antigen probe for detecting class-specific antibodies to the HCV core in patients with acute and chronic hepatitis C and for simultaneous detection of antibodies to HBV core and those to HCV core in donated blood.

Hepatitis C virus (HCV) samples in 155 sera, from patients with chronic non-A, non-B liver disease and blood donors, were grouped into four genotypes (I, II, III, and IV) by amplification of core-gene sequences by polymerase chain reaction with type-specific primers. HCV genotypes were compared with various HCV-associated antibodies detectable by the first-generation ELISA (ELISA-1) with C100-3 protein and a second-generation immunoblot assay with four recombinant HCV proteins. Antibodies to C100-3 protein and those to its subsequence (5-1-1) were detected in 13 (93%) and 12 (86%), respectively, of 14 sera with genotype I HCV; 56 (79%) and 58 (82%) of 71 sera with genotype II; 13 (34%) and 6 (16%) of 38 sera with genotype III; and II (34%) and 4 (13%) of 32 sera with genotype IV. Amino acid sequences of C100-3 of genotype I HCV are conserved by approximately 90% in genotype II, but only by approximately 75% in genotypes III and IV. The sensitivity of ELISA-1, therefore, would be influenced by heterogeneity in C100-3 sequences of different genotypes.

China has not been extensively investigated for the prevalence of hepatitis C virus (HCV) infection among people with or without liver disease. We analyzed serum from 2,177 liver disease patients from 7 cities in different areas of China. Of 435 acute hepatitis patients, only 11% were positive for HCV RNA, while hepatitis B surface antigen (HBsAg) was detected in 33%. Of 1,668 patients with chronic liver disease, 14% and 74% were positive for HCV RNA and HBsAg, respectively. Nearly 80% of non-B chronic liver disease were negative for HCV RNA. The frequency of HCV RNA in chronic liver disease was significantly higher in Hami (32%) and Shenyang (30%) than in other cities (6-12%). The HCV genotype distribution varied by region. Genotype III was detected in 46-70% of HCV infections in Hami, Shenyang, and Lanzhou, while more than 90% of patients from southern cities (Nanjing, Nanning, and Chengdu) had genotype II. No evidence for genotype I or IV infections was found. A full-length HCV genome sequence (HC-C2) derived from a Beijing patient with genotype II was closely related to previous isolates from Japanese and Taiwanese patients. These results suggest that HCV prevalence and genotype distribution vary from region to region in China, and that the HCV now predominant in China may have evolved epidemiologically with infections in Japan and Taiwan. The study identified a high frequency of non-B, non-C chronic liver disease in China, suggesting possibly a new agent or infections with extreme variants of HCV.

Cultured hepatoma cells (HepG2) were cotransfected with two different plasmids carrying a head-to-tail dimer of recombinant hepatitis B virus (HBV) DNA cloned from deletion mutants isolated from the circulation of persistently infected hosts. They were tested for the secretion of viral particles with mutant genome encapsidation. A recombinant plasmid defective in the S gene and one defective in both the C and P genes complemented in trans for the production of viral particles. Mutant genomes from either of the recombinants were encapsidated. Similarly, a recombinant defective in the C gene and another defective in the P gene transcomplemented for the production of viral particles containing mutant genomes. A hepatoma cell line with integrated HBV DNA sequences defective in the C and P genes (PLC/PRF/5) when transfected with a recombinant defective in the S gene produced viral particles with the HBV genome from the transfecting recombinants. These results confirm the expected transcomplementation among the S, C and P genes of HBV, when either episomal or integrated into chromosomes, for the maintenance of defective HBV mutants in persistently infected hosts.

A monoclonal antibody (1-18) was raised against an enneapeptide representing amino acids 125 to 133 of the product of the S gene of hepatitis B virus DNA [S(125-133) segment] with a sequence of Thr-Ile-126-Pro-Ala-Gln-Gly-Thr-Ser-Met. Another monoclonal antibody (T-7) was raised against an S(125-133) segment in which Ile-126 was replaced by Thr-126. In a panel of 16 samples of hepatitis B surface antigen (HBsAg) with known S gene sequences, 1-18 reacted with 5 with Ile-126. T-7 reacted with 10 HBsAg samples with Thr-126; it did not, however, react with the remaining one of subtype ayw with Thr-126 flanked by Met-125 and Thr-127. The two allelic subtypic determinants, specified by Ile-126 and Thr-126 and distinct from d/y or w/r, were named i and t after isoleucine and threonine, which regulate them. They were expressed in a mutually exclusive fashion in 216 (83%) of 260 HBsAg samples from asymptomatic carriers. They were not detected in 36 (14%) samples; the failure to detect an i or t determinant was particularly common in HBsAg samples of subtype ayw (26 [79%] of 33). A part of the S gene sequence was determined for eight HBsAg samples without a detectable i or t determinant. They had an Ile-126 or Thr-126 residue that was flanked by Thr-127, not the Pro-127 commonly possessed by HBsAg samples displaying an i or t determinant. Expression of the i/t allele, therefore, would require Pro-127. In eight (3%) of the samples, both i and t determinants were detected; the presence of i and t on the selfsame HBsAg particles was verified by sandwiching the particles between 1-18 and T-7. A point mutation from thymine to cytosine at nucleotide 377 in the S gene, contributing different second letters to codon 126 (ATT for Ile and ACT for Thr), would have been responsible for the assembly of HBsAg particles with both i and t determinants by means of phenotypic mixing.

In November 1989, Japanese Red Cross Blood Centres started screening for heaptitis C virus (HCV) with enzyme-linked immunosorbent assay (Elisa) for the C100-3 viral peptide as the first such nationwide programme in the world. Thereafter post-transfusion non-A non-B hepatitis (PTNANBH) was reduced by 61-80%, but this was not as complete a success as our programme to prevent post-transfusion hepatitis B by screening for high titer hepatitis B core antibody, which we began in the same period. In order to acquire more effective control of PTNANBH, the HCV core-related antigen (GOR, N14) and second-generation Elisa (Ortho2, Abbott2)and second-generation antigen agglutination (PA, PHA) tests have been employed. Among 16,500 donors in 11 blood centers, 365 were serologically positive by at least one of these tests. Among these, HCV RNA was detected in 138 units and the remaining 227 were HCV RNA negatives. The effectiveness of these serological tests to detect HCV RNA-positive status were analyzed. Passive haemagglutination and particle agglutination (PHA and PA) tests were highly effective to predict HCV viraemia among blood donors. Also, these tests can easily determine antibody titre. By either PHA or PA, all units with greater-than-or-equal-to 2(12) agglutination titre (120 and 122 units) were HCV RNA positive and all agglutination-positive units with serum alanine aminotransferase level higher than 35 Karmen units were HCV RNA positive. These results have formed the basis for implementing a more effective screening for HCV viraemia in blood donors, where effectiveness is defined as enhancing the protection of patients from post-transfusion hepatitis C and providing higher quality information to achieve more effective donor counselling.

A method was developed for the analysis of heterogeneity in antigen polypeptides in individual sera. Polypeptides in sera were adsorbed by polystyrene beads coated with antibody in wells of a microplate. They were dissociated with a small volume of elutant, and transferred to slots on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Polypeptides separated on gel were then immunoblotted with antibodies labeled with horseradish peroxidase The method was applied to analyze different populations of hepatitis B surface and e antigen polypeptides in sera from carriers of hepatitis B virus. Applicability to mass-scale and high sensitivity of the method would allow surveys of heterogeneous antigen polypeptides in serum for any biological significance

Anti-hepatitis C virus (HCV) core antibodies in blood donors were detected in enzyme-linked immunosorbent assay with oligopeptides (CP9 and CP10) corresponding to the putative core protein of HCV and their performances were compared with C100-3 antibodies. HCV-RNA was detected by nested polymerase chain reaction in fresh plasma samples from blood donors positive for anti-core antibodies in hihg-titers and/or anti-C100-3 antibodies. In plasma samples from 2, 034 apparently healthy blood donors, anti-CP9 was detected in 133 (6.5%), anti-CP10 was detected in 95 donors (4.7%) and anti-C100-3 was detected in 33 donors (1.6%). Among plasmas positive by at least one test, 31 were found to contain HCV-RNA; 19 of them were negative for anti-C100-3, but positive for anti-CP9 and/or anti-CP10 in high titers (OD≥1.000). These results indicated that anti-core antibodies would be useful in complementing anti-C100-3 for further decreasing the incidence of post-transfusion non-A, non-B hepatitis.

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 c 100-3 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 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.

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.

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.