岡本 宏明

An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes Via and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype IV/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 1d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a.

Objectives: Mutations in the precore region are not always detected in hepatitis B virus (HBV) from patients with chronic hepatitis B who respond to interferon and lose hepatitis B e antigen (HBeAg) from serum, The other mutations may also be responsible for the loss of HBeAg and response, Methods: Forty six consecutive patients with HBeAg-positive chronic hepatitis B received recombinant-alpha 2a interferon (total dose: 702 MU), The mutation for stop codon 28 in the precore region nas determined by restriction fragment length polymorphism, and mutations in the core promoter were searched for in five HBV DNA clones propagated from each patient, Results: HBeAg was cleared at 6 months after interferon in 11 (61%) of 18 patients with the precore mutation and in 12 (43%) of 28 without it, Of these 28 patients, 19 with mutations in the core promoter in all five HBV DNA clones lost HBeAg more frequently than the remaining nine who had at least one clone among the five that lacked such mutations (58 vs 11%, p < 0.05), Conclusions: HBeAg-positive patients infected with HBV variants having mutations for an HBeAg-negative phenotype would respond better to interferon by clearing HBeAg from serum, Such mutations may not necessarily be in the precore region but also in the core promoter, which would interfere with the synthesis and secretion of HBeAg either at the translation or transcription level.

Some HLA class II alleles and haplotypes were examined by restriction fragment length polymorphism of corresponding DNA fragments amplified by the polymerase chain reaction in 117 patients with chronic hepatitis C in Japan. The prevalence rates were compared between patients and 1216 controls and in 67 patients with liver cirrhosis, of whom 20 had hepatocellular carcinoma and 50 patients with ch ron ic hepatitis who did not have cirrhosis or hepatocellular carcinoma. Notably, DRB1*0405 (49% [95% confidence range 38-60%] vs. 26% [16-40%]; P < 0.05, relative risk [rr] = 2.8) and DOB1*0401 (43% [33-54%] vs. 22% [13-34%]; P < 0.05, rr = 2.1) were detected more frequently in patients with cirrhosis than in those without cirrhosis. By contrast, DRB1*0901 (11% [6-19%] vs. 28% [18-40%]; P < 0.05; rr = 0.3) and DQB1*0303 (11% [6-19%] vs. 36% [25-49%]; P < 0.01; rr = 0.2) were detected less frequently in patients with cirrhosis than those without cirrhosis. Accordingly, the DRB1*0405-DQB1*0401 haplotype was more common (43% [33-54%] vs. 22% [13-34%]; P < 0.05; rr = 2.7), while the DRB1*0901-DQB1*0303 haplotype was less common (9% [4-17%] vs. 28% [18-40%]; P < 0.05; rr = 0.3) in patients with cirrhosis than in those without cirrhosis. These results suggest that there would be HLA class II alleles and haplotypes which may be associated with an accelerated or slower progression of chronic hepatitis C towards cirrhosis and eventually to hepatocellular carcinoma. (C) 1996 Wiley-Liss, Inc.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124-147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130-132 of the S-gene product, In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV, One of the three clones from another patient had Ser-126 in place of lie or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

RNA of a non-A to E hepatitis virus identified recently and designated provisionally GB virus C (GBV-C), was sought in patients in Indonesia by reverse-transcription polymerase chain reaction with nested primers deduced from a helicase-like region. GBV-C RNA was detected in 32 (55%) of 58 patients on maintenance hemodialysis at a frequency significantly higher (P < 0.001) than that in seven (5%) of 149 patients with chronic liver disease, Go-infection with hepatitis C virus was observed in 26 (81%) of the 32 patients on hemodialysis and in five (71%) of the seven patients with liver disease who were infected with GBV-C. Complete identity was observed in a sequence of 100 base pairs in the helicase-like region for GBV-C cDNA clones from some patients on maintenance hemodialysis. These results indicate that the patients on hemodialysis would be at high risk for GBV-C infection, which would be transmitted by transfusion and patient-to-patient routes. (C) 1996 Wiley-Liss, Inc.

Background. A recently discovered non-A-E hepatitis virus has been designated hepatitis GB virus C (HGBV-C), but little is known about its mode of transmission and its clinical manifestations. We studied 519 patients on maintenance hemodialysis to determine whether they were infected with HGBV- C. Methods. HGBV-C RNA was identified in serum by a reverse-transcription- polymerase-chain-reaction assay with nested primers deduced from a nonstructural region. A nucleotide sequence of 100 bp in the nonstructural region was determined on HGBV-C clones. Results. HGBV-C RNA was detected in 3.1 percent of the patients on hemodialysis (16 of 519), as compared with 0.9 percent of healthy blood donors (4 of 448, P&lt 0.03). None of the 16 patients had evidence of active liver disease, although 7 were also infected with hepatitis C virus. Eight patients with HGBV-C infection were followed for 7 to 16 years. In two patients the virus was present at the start of hemodialysis. One had a history of transfusion, and HGBV-G RNA persisted over a period of 16 years the other became free of HGBV-C after 10 years. In five patients, HGBV-C RNA was first detected 3 to 20 weeks after blood transfusion and persisted for up to 13 years. One patient with no history of transfusion was infected with an HGBV-C variant with the same sequence as in two of the patients with post-transfusion HGBV-C infections. Conclusions. Patients on maintenance hemodialysis are at increased risk for HGBV-C infection. This virus produces persistent infections, which may be transmitted by transfusions but may also be transmitted by other means.

The detection of hepatitis C virus (HCV) in blood donors and patients with acute and chronic hepatitis has brought to the fore another virus or viruses which can be transmitted parenterally and induced liver disease. The RNA of a candidate virus designated GB virus C (GBV-C) was determined by the polymerase chain reaction with primers deduced from a helicase-like region in 229 leprous patients in Japan. GBV-C RNA was detected in 12 (5.2%) patients, and HCV RNA in 41 (18%). Three patients were coinfected with GBV-C and HCV. The nine patients infected with GBV-C alone had aminotransferase levels lower than the three patients with the mixed infection or the 38 patients infected with HCV only (P&lt 0.001). Sequence comparison within 100 base pairs in the helicase-like region suggested that two, three and three patients, respectively, would have been infected with three distinct strains of GBV-C. These results indicate that patients with leprosy are at increased risk for infection not only with HCV, but also with GBV-C, and that the infection with GBV-C alone would not induce hepatic injuries as severe as HCV infection.

The cold activation of complement, determined by hemolytic activity of sera stored at low temperature, closely correlates with ongoing hepatitis C virus (HCV) infection. Of 53 189 blood units donated at a blood center in Japan, 187 (0.35%) were positive for antibody to HCV of which 65 (35% of the units with antibody) contained detectable HCV RNA. While sera from the 65 units with HCV RNA had been stored at 4 degrees C, the cold activation of complement was observed in 43 (66%) at day 1, an additional 13 (20%) at day 4, and further in 4 (6%) at day 7. Thus, the cold activation was observed in 60 (92%) of 65 sera with HCV RNA during the storage at low temperature for 7 days. Cryoglobulins were detected only in 13 of the 43 sera which showed the cold activation at day 1, and the donors with cryoglobulinemia were older than the 52 viremic donors without it (mean +/- S.D.: 47 +/- 10 vs. 40 +/- 11 years, P < 0.05). There were no differences in the distribution of HCV genotypes in sera which showed the cold activation at day 1; they were 24 (69%) of 35 sera with genotype II/1b, 15 (68%) of 22 with III/2a and 4 (50%) of 8 with IV/2b. The results indicate that it would take up to a week before the cold activation is fully manifested in sera from persons infected with HCV.

Background/Methods: Markers of hepatitis C virus infection were tested for in 229 patients with leprosy (male 154, female 75) in Japan. Results: Antibody to hepatitis C virus by a second-generation enzyme immunoassay was detected in 68 patients (30%), and RNA of hepatitis C virus in 41 (18%), in prevalence rates much higher (p<0.001) than those in matched controls (11/923 or 1.2% and 9/923 or 1.0%, respectively), Hepatitis C virus genotypes were II/1b in 37 (90%), III/2a in three (7%) and IV/2b in one (2%), in which II/1b was more frequently (p<0.003) represented than in hepatitis C virus carriers without leprosy in Japan (520/767 or 68%), The 41 patients with hepatitis C virus viremia had serum transaminase levels significantly higher than those in the other 188 patients without viremia (p<0.01). Conclusions: These results indicate that leprous patients confined in institutions are at high risk of hepatitis C virus infection, and that patients infected with hepatitis C virus should be monitored for liver function and placed on interferon therapy whenever required.

Hepatitis C virus (HCV) RNA was tested for, and HCV genotypes determined, in 96 patients with haemophilia A in japan. Of 88 patients aged greater than or equal to 10 years, 74 (84%) were positive for HCV RNA at a frequency higher than that in patients aged less than 10 years (one of eight, 13%, P<0.001). Genotype I/1a was detected in 30 (40%), II/1b in 12 (16%), III/2a in eight (11%), IV/2b in five (7%) and V/3a in 12 (16%); mixed infection with HCV of two different genotypes was identified in the remaining nine (12%), This distribution was markedly different from that in 767 Japanese HCV carriers without haemophilia, in whom II/1b accounted for the majority (68.7%), I/1a was rare (0.5%), V/3a was absent, and mixed infection was observed rarely (1.3%), Mixed infection was transient in all of the seven haemophilic patients who were followed for 1 to 7 years, One of them was infected with genotype II/1b and an unclassifiable genotype, which showed nucleotide sequence similarity to genotype 4c from Zaire (82% homology in the El gene) and to 4a from Egypt (91% homology in a part of the NS5b region), In this patient, HCV of genotype II/1b disappeared while that of group 4 survived during a 4-year observation period, These results indicate different epidemiology of HCV genotypes in Japanese haemophiliacs, attributable to HCV contaminating factor VIII imported in the past, and an increased opportunity in haemophiliacs for mixed infection with HCV of different genotypes.

A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e,, I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype Ic in Simmond's classification.

Objectives: Because of its specific association with hepatitis C virus (HCV) infection, the cold activation of complement is an easy and inexpensive indicator of HCV viremia, It was evaluated for eligibility as a marker of response to interferon in patients with hepatitis C, Methods: The cold activation of complement was determined by the loss or decrease of hemolytic activity with the microtitration method in sera that had been stored at 4 degrees C overnight. Results: We observed the loss of hemolytic activity by the cold activation of complement in 236 (72%) and a decrease in 56 (17%) of 327 sera from patients with HCV-associated chronic liver disease, which was much more (p < 0.001) than in 1 (1%) and 13 (14%), respectively, of 94 sera from patients with chronic liver disease associated with hepatitis B virus infection, Interferon-alpha (total dose 516 x 10(6) units) or interferon-alpha 2b (774 x 10(6) units) was given to 67 patients with chronic hepatitis C, of whom 56 had the cold activation of complement, The response to interferon was evaluated by the clearance of serum HCV RNA at 6 months after the completion of therapy, The cold activation of complement disappeared in 18 patients, of whom 15 (86%) responded, It persisted or fluctuated in the remaining 38 patients, only six (16%) of whom responded to interferon (p < 0.001), The cold activation of complement once disappeared at the completion of interferon and then reappeared in patients who relapsed after completing interferon therapy, Conclusions: These results indicate that the cold activation of complement may be associated with the presence of HCV in blood and a lower rate of durable response after completion of interferon therapy.

Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279-9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups.

We tested hepatitis C virus (HCV) antibody in 4216 sera collected fi-om healthy people living in European part of Russia (including Northern, North-Western, Central, Central-Blacksoil, Volga-Vyatka, Volga, and North-Caucasian regions), non-European part of Russia (the Urals, East-Siberia, and the Far-East regions) and Mongolia. Prevalence of HCV antibody varied significantly by regions, ranging from 0.7% in Central region of European part of Russia to 10.7% in Mongolia. Genotyping of HCV (into 1a, 1b, 2a, 2b, and 3a) was performed on 469 sera from blood donors and patients (in Russia, Moldova, Turkmenistan, and Mongolia) who were positive for both HCV antibody and RNA. Genotype 1b was the most dominant genotype irrespective of regions (68.9%), with the highest rate in Moldova (96%). HCV unclassifiable into genotypes 1a-to-3a was found in 28 (6.0%) samples: particularly 4 of 10 samples from Lipetzk were untypable. Overall, HCV genotypes in European part of Russia were more similar to those in European countries, while those in Eastern part of Russia more similar to China or Japan. Genotype distribution was not associated with the clinical expression of HCV disease: acute hepatitis, chronic hepatitis or liver cirrhosis.

We sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.

Human leukocyte antigen B44-restricted cytotoxic T lymphocytes (CTLs) recognize an epitope in hepatitis C virus (HCV) nucleoprotein residues 81-100. CTLs that recognize two wild-type peptides 81-100 of HCV genotypes 1b/II and 2a/III were generated from peripheral blood lymphocytes of each of three patients studied. Although CTLs that recognize a wild-type peptide 81-100 of HCV genotypes 1a/I and 2b/IV were not generated from any patient, CTLs that recognize peptide 81-100 of a rare HCV isolate of type 1a/I were generated from two patients. The results suggest that HLA B44-restricted CTLs recognize most, if not all, HCV isolates of types 1b/II and 2a/III and rare variants of type 1a/I and that the wild-type HCV isolates of genotypes 1a/I and 2b/IV may be less immunogenic for HLA B44-restricted CTLs.

Amino acid residues 81-100 of the hepatitis C virus (HCV) nucleoprotein contain a cytotoxic T cell epitope that is recognized by cytotoxic T lymphocytes (CTLs) in association with human leukocyte antigen B44. With panels of truncated and overlapping peptides, the minimal and optimal epitope recognized by CTLs was shown to be a 9-mer peptide (residues 88-96). The peptide can stimulate effectively CTLs that are able to recognize endogenously synthesized and processed HCV nucleoprotein.

From September 9 to October 3, 1994, five patients on maintenance hemodialysis in a dialysis unit in Tokyo contracted hepatitis B virus (HBV) infection successively, and four of them died of fulminant hepatitis. The unit treated 181 patients three times a week on eight shifts, and all five afflicted patients were on the same shift along with 27 other patients. HBV DNA clones from the hepatitis patients had a point mutation converting codon 28 in the precore region to a stop codon, which aborts the synthesis and secretion of hepatitis B e antigen, and showed a sequence similarity of >99.5% within 645 base pairs covering the X gene and precore region. There were two HBV carriers with antibody to hepatitis B e antigen who were receiving hemodialysis on the same shift. HBV DNA clones from one of them had the stop codon 28 in the precore region, and a sequence similarity of >99.7% to those from the five patients. Based on these results, it was deduced that the fulminant HBV strain was transmitted from the carrier to five patients, and resulted in the death of four. The outbreak indicates that immunocompromised hosts like hemodialysis patients can develop fulminant hepatitis B if and when they are infected with extremely virulent HBV strains.

Sera from 49,088 blood donors were tested for markers of hepatitis C virus (HCV) infection and decreased hemolytic activity after they had been stored at 4 degrees C for 24 h, a phenomenon known as the cold activation of complement. Antibody to HCV (anti-HCV) was detected in 315 (0.64%) units, of which 181 (57%) were positive for HCV RNA. The cold activation of complement was detected in 170 (0.35%) units, and HCV RNA was detected in 140 (82%) of them. Thus, the cold activation of complement was observed in 140 (77%) of 181 blood units with HCV RNA. The close association of HCV viremia with the cold activation of complement would be useful as a surrogate test in preventing post-transfusion HCV infection in developing areas where anti-HCV assays are not easily performed.

The effect of recombinant interferon-alfa on serum HCV RNA levels in Japanese patients with chronic hepatitis C was investigated, At 24 weeks of treatment, 41 (32.5%) of 126 patients lost HCV RNA from serum, and aminotransferases were normalized in 31 (75.6%) of these 41 cases. HCV genotypes were categorized into four types (Type I, II, III, IV); the frequencies among the patients were: Type I: 0%, Type II: 70.6%, Type III: 20.6%, and Type IV: 6.3%. At the end of the 24-week treatment, HCV RNA levels were remarkably decreased in Type III patients and became undetectable in 18 (69.2%) of 26. In contrast, only 18 (20.2%) of 89 patients with Type II and two of eight with Type IV lost HCV RNA from sera. The relation between HCV genotype (Type III) and response to IFN therapy was also confirmed using a logistic regression model. HCV genotype seems to be an important factor in determining the response to IFN in patients with chronic hepatitis C.

Background/Aims: Several genotypes of hepatitis C virus (HCV) have been identified by phylogenetic analysis, but their clinical relevance remains elusive, Liver transplantation for HCV-related cirrhosis offers a unique opportunity for prospective studies of this issue, Methods: Sixty anti-HCV-positive liver recipients with precise virological, and histological assessments were included, HCV genotype was determined with both type-specific capsid primers and a line probe genotyping assay. Results: HCV genotype Ib was the predominant type before transplantation (40 of 60 patients); after liver transplantation, acute and chronic active hepatitis developed more frequently in these patients than in patients infected by other genotypes (31 of 40 and 24 of 40 vs. 8 of 20 and 4 of 20 patients), Actuarial rates of acute hepatitis and chronic active hepatitis were 77% and 59%, respectively, 3 years after transplantation in patients infected by type Ib and 40% (P = 0.008) and 22% (P = 0.004) in those infected by other types, There was no statistical relation between the level of MCV viremia and HCV genotypes both before and after transplantation, in contrast, after transplantation, serum HCV RNA Values were: significantly increased in patients who developed hepatitis after transplantation. Conclusions: This study provides direct evidence that HCV Ib is associated with more aggressive recurrent liver disease than other genotypes.