岡本 宏明

We analyzed the surface epitopes of hepatitis B virus (HBV) virions with two types of vaccine-escape mutations within the S gene (S126 and S145) by in vitro neutralization study using affinity chromatography. Each of the two epitopes of the S gene product (recognized by monoclonal antibodies Nos. 3207 and 7604) was lost significantly from the surface of HBV virions with the S145 or S126 mutation. However, the preS2 epitope recognized by the No. 5520 monoclonal was preserved on the virion surface of both mutants. Our results, thus, suggest that a preS2-containing vaccine could possibly prevent infections with HBV S gene mutants that escape from or evolve after immunization with conventional vaccine and hepatitis B immune globulin (HBIG).

Three hepatitis C virus (HCV) isolates were obtained from patients with chronic liver diseases in Indonesia which were not classifiable into any of the genotypes I/1a, II/1b, III/2a, IV/2b or V/3a reported previously. The entire nucleotide sequence was determined for one HCV isolate (HC-G9); the remaining two isolates were of the same genotype based on a > 95% similarity within their partial sequences spanning 2927 nucleotides (nt). The HC-G9 genome consisted of 9440 nt including the 5' untranslated region of 341 nt, an open reading frame of 9033 nt coding for a polyprotein of 3011 amino acids and the 3' untranslated region of 66 nt (U stretch of 17 to 47 nt at the extreme 3' terminus excluded). It differed by 20 to 33% in nucleotide sequence from any of 14 HCV genomes of genotypes I/1a to IV/2b whose full-length sequences are known. By the unweighted pair-group method with arithmetic mean, HC-G9 was on a major branch (group 1) of the phylogenetic tree of HCV to which genotypes I/1a and II/1b belong. It is proposed, therefore, that the novel genotype for HC-G9 should be called 1c. A method was developed to identify genotype 1c by PCR with a primer deduced from the core gene that was specific to it. Since genotype 1c was detected in seven (15%) of 48 HCV RNA samples from Indonesian patients with chronic liver disease, but not in any of 1097 from other districts of the world, it appears to have evolved and remained in Indonesia. In addition to its epidemiological importance, the association of genotype 1c HCV with the severity of liver disease and its response to interferons deserve to be evaluated.

Background. Although there are case reports of vertical transmission of hepatitis C virus (HCV), it remains uncertain to what extent infected mothers transmit this virus to their infants. Methods. We investigated the transmission of HCV from infected mothers to their babies by analyzing HCV RNA in the blood. Three independent studies were performed. First, 7698 parturient women were tested for anti-HCV antibodies; 53 were positive. Their 54 infants (including one set of twins) were followed prospectively for at least six months and tested for HCV infection. Second, the babies of six women with known HCV disease were prospectively studied. Third, the families of three HCV-infected infants were examined retrospectively. Results. Of the 53 antibody-positive mothers, 31 were also positive for serum HCV RNA, Three of the 54 babies born to these mothers (5.6 percent) became positive for HCV RNA during the follow-up period. None of the babies of the 22 women who were antibody-positive but HCV RNA-negative became positive for HCV RNA. In the second study, HCV RNA was detected in one of the six infants of infected mothers. In the third study, HCV RNA was detected in the mothers of the three HCV-infected infants. In each of the seven infected infants we studied, the genomic sequence of HCV was almost identical to that from the mother. These seven mothers had significantly higher titers of HCV RNA than did the mothers of infants with no evidence of infection (mean [+/-SD], 10(6.4+/-0.5) VS. 10(4.4+/-1.5) per milliliter; P<0.001). Conclusions. HCV is vertically transmitted from mother to infant, and the risk of transmission is correlated with the titer of HCV RNA in the mother.

Blood units from 3383 donors were tested for antibodies to hepatitis C virus (HCV) by a second-generation passive hemagglutination assay (PHA-2) with detector cells coated with three recombinant HCV proteins. They were tested also for antibodies to synthetic HCV core peptides (anti-CP9 and anti-CP10) by enzyme immunoassays and for alanine aminotransferase (ALT). PHA-2 was reactive in 32 (0.9%) units, high-titered anti-CP9 and/or anti-CP10 was detected in 101 (3.0%), and elevated ALT levels (>45IU/L) in 99 (2.9%). At least one of these markers were detected in 209 units (6.2%), and HCV RNA was tested for in 197 of them. HCV RNA was detected in 21 units including 19 initially reactive on PHA-2 of which 17 were repeatedly reactive, 19 with anti-CP9 and/or anti-CP10 in high titers, and 5 with elevated ALT levels. Two units were reactive only on PHA-2, while the other two had anti-CP9/anti-CP10 as the sole indicator of hepatitis C viremia. These results indicated that PHA-2 and anti-CP9/anti-CP10 would be complementary in detecting blood units with HCV RNA, and effective in further decreasing the risk of post-transfusion hepatitis C.

Antibodies to the hepatitis C virus (HCV) core of various immunoglobulin classes were determined by enzyme immunoassays with three synthetic peptides, CP14 (amino acids 5-40 of the core protein), CP10 (5-23), and CP9 (39-74). in 735 patients with chronic type C liver disease, anti-CP14, anti-CP10, and anti-CPS of IgG class were detected in 99%, 94%, 82%, respectively; those of IgM class in 86%, 69%, and 39%; and those of IgA class in 56%, 40%, and 4%. Thus anti-CP14 was more prevalent than anti-CP10 or anti-CPS in every immunoglobulin class. The prevalence of IgM anti-CP14 was much higher (P < 0.001) in patients (116/135 or 86%) than in asymptomatic carriers of HCV (13/39 or 33%). In seven patients with acute hepatitis C, IgM anti-CP14 continued to decrease in two in whom hepatitis resolved, but increased in five in whom hepatitis once resolved and then exacerbated. IgM anti-CP14 was followed in 30 patients with chronic hepatitis C during 24 weeks while they received recombinant interferon alpha-2a. IgM anti-CP14 decreased remarkably within 8 weeks in all of them. Thereafter, it continued to decrease in nine patients who responded to interferon and lost HCV RNA from circulation, but started to increase in five non-responders who continued to have high titers of HCV RNA, In the remaining 16 patients in whom HCV RNA decreased once and then increased, IgM anti-CP14 continued to decrease till 20 weeks and then increased. These results indicate that IgM anti-CP14 reflects the activity of liver disease, and is useful in following the outcome of patients with acute hepatitis C and in monitoring the response to interferon in patients with chronic hepatitis C. (C) 1994 Wiley-Liss, Inc.

Interferon induces remission in about 50% of patients with chronic hepatitis C, but it is difficult to predict which patients will respond. Host and viral factors were evaluated for correlation with response to interferon in patients with chronic hepatitis C. Recombinant interferon alpha-2b with a total dose of 480-560 million units was given to 136 patients, of whom 74 (54%) responded. Genotypes of hepatitis C virus (HCV) in sera, I, II, III, IV, and V, were determined by polymerase chain reaction (PCR) with type-specific primers. In 72 patients, pretreatment levels of HCV RNA were titrated by PCR in serial tenfold dilutions of RNA extracted from serum. Response to interferon occurred in 34 (40%) of 85 patients infected with HCV of genotype II, less frequently than in 22 (85%) of 26 with genotype III (P < 0.001) or in 7 (70%) of 10 with genotype IV. Of 51 patients with genotype II HCV, 6 of 8 (75%) with HCV RNA titers (10(6) responded, more frequently than 4 of 43 (9%) with titers greater than or equal to 10(6) (P ( 0.001). Responders were younger than non-responders (45.7 +/- 11.7 vs. 50.3 +/- 9.6 yr) and had received transfusions less frequently (26/74 or 35% vs. 37/62 or 60%, P < 0.01). Response to interferon correlated inversely with the severity of liver histopathology. These results indicate that response to interferon is influenced by HCV genotypes and pretreatment levels of HCV RNA in serum. (C) 1994 Wiley-Liss, Inc.

Background: Hepatitis C virus infections are known to be common in injectable drug users (IDU) both in New Zealand and overseas. Little is known of the hepatitis C genotype frequency in this population. Aims: To confirm the high incidence of hepatitis C virus infections in IDU and compare this with the frequency in oral drug users (ODU) as well as identify the pattern of hepatitis C genotypes present.

SUMMARY. Patients on maintenance haemodialysis in four dialysis centres were tested for markers of hepatitis C virus (HCV) infection. Antibody to HCV (anti‐HCV) was detected by the second‐generation enzyme immunoassay in 142 (26%) of the 543 patients and HCV RNA in 117 (22%) of whom four were without detectable anti‐HCV in serum. Seventy‐seven (66%) were infected with HCV of genotype II/1b, 31 (27%) with genotype III/2a and eight (7%) with genotype IV/2b. in a distribution similar to that in blood donors who carried HCV asymptomatically. Haemodialysis patients had high HCV RNA titres comparable to those of patients with chronic hepatitis C. HCV RNA was detected in 96 (26%) of the 365 patients with a history of transfusion more frequently than in 21 (12%) of the 178 without previous transfusion (P&lt 0.001). In transfused patients, frequencies of anti‐HCV and HCV RNA increased in parallel with the duration of haemodialysis. The frequency of anti‐HCV in non‐transfused patients, however, did not change appreciably with the duration of haemodialysis up to 22 years. The patients with anti‐HCV had a higher frequency of HCV RNA in serum than symptom‐free blood donors with anti‐HCV (113/142 or 80%vs 109/166 or 66%P&lt 0.01) and the patients with HCV RNA had a lower frequency of elevated aminotransferase levels than blood donors with HCV RNA (5/113 or 4%vs 27/109 or 25%, P&lt 0.00l). These results indicate that transfusion is a significant cause of HCV infection in patients on maintenance haemodialysis, and that these patients are prone to establish the HCV carrier state after infection. Copyright © 1994, Wiley Blackwell. All rights reserved

We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5' untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1 a, lb, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3' end of 5' UTR, C, El and 5' end of E2/NS1) and 8276 to 9394 (3' end of NS5 and 3' UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96 %, compared to 73 to 74 %, 73 %, 70 % or 69 to 70 % against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported 'type V (3a)' isolates was 88 to 100 % ; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3' UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5'CGTAAAACTTCT GAACGGTC, sense and no. 339: 5'GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.

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.