岡本 宏明

Hepatitis C virus (HCV) is the major aetiological agent for blood borne non-A, non-B hepatitis worldwide, Since its discovery in 1989, at least 28 HCV genotypes have been reported, which differ by >20% in the nucleotide sequence of the entire genome (similar to 9500 nucleotides) or the sequence of the E1 gene (576 nucleotides). Different HCV genotypes have distinct geographical distributions, and may be associated with variations in viral replication and disease-inducing activity, as well as poor response to interferons in patients with chronic hepatitis C.

We found a rare hepatitis C virus (HCV) genotype in 11 Italian and three French patients. The HCV core gene sequence for these patients showed nucleotide similarities of 93-98% to each other, 88-92% to genotypes III (2a) and IV (2b) isolates, and only 82-85% to genotypes I (1a), II (1b), 1c, and V (3a) isolates. The NS5b sequence also indicated the distinctness of this group of HCV, its relatively close relationship to genotype III (2a) and IV (2b), and a high degree of similarity to several isolates provisionally classified as '2c'. The El region sequence determined in one French patient also had a high similarity to another '2c' isolate reported previously. Although the prevalence of this '2c' HCV in Italian patients was low (11/173: 6%), it was of clinical interest. This genotype was associated with poor responsiveness to interferon therapy and also with poor antibody response to C100-3 antigen, and two of the 11 Italian patients had mixed cryoglobulinemia, Interestingly, all the three French patients with the '2c' HCV were from Italy, and virtually Italians.

Some of the reported hepatitis B virus (HBV) sequences have an additional in-frame initiation codon, ATG, 56 nucleotide triplets upstream of the X gene's ATG. Between the two ATGs, an in-frame termination codon exists in some isolates but not in all that have been reported. To investigate the possibility that this presumed 'preX' region is expressed, we examined 24 healthy carriers and 33 chronic liver disease patients for the 'preX' nucleotide sequence, Results indicated that 5 of the 24 healthy carriers and 21 of the 33 patients (21% vs, 64%, P < 0.003) had this 'preX' region as an open reading frame (ORF). Moreover, there was a unique set of mutations that could be found frequently in patients but not in healthy carriers, suggesting a disease-implicated HBV 'preX' variant. These mutations were associated with amino acid substitutions in the 'preX' ORF but not in the polymerase ORF. These results suggest the importance of mutations in the 'preX' region for the pathogenesis of HBV, although with yet unclear mechanisms.

Objective: Fulminant hepatitis B can be induced by hepatitis B virus (HBV) strains with mutations in the precore region that cannot encode hepatitis B e antigen (HBeAg). Such mutations are rarely seen in HBV DNA clones from patients with fulminant hepatitis B in the United States and France. Thus, the other mutations in HBV strains causing fulminant hepatitis B need to be identified. Design: Retrospective clinical, serologic, and molecular biological studies of patients with fulminant hepatitis B. Setting: University and city hospitals in Japan. Patients: 43 patients with fulminant hepatitis B. Measurements: The precore region coding fora part of the HBeAg precursor and the core promoter regulating the transcription of precore messenger RNA were sequenced in HBV DNA clones. Results: A point mutation from G to A at nucleotide 1896 in the precore region was detected in 519 (98%) of 529 HBV DNA clones from 38 patients. Two point mutations in the core promoter, from A to T at nucleotide 1762 and from G to A at nucleotide 1764, were detected in all 130 clones from the remaining 5 patients, who did not have mutations in the precore region, and in 20 (63%) of 32 clones from a patient with chronic hepatitis B who had transmitted HBV to 1 of these other 5 patients. Mutations in the core promoter were also detected in clones from 26 (68%) of the 38 patients with the precore mutation at nucleotide 1896. Neither HBeAg nor antibody to HBeAg was detected in 37 (90%) of the 41 patients tested. Conclusions: In Japan, fulminant hepatitis B is closely associated with HBV strains that do not produce HBeAg because of mutations in the precore region, which affect translation of HBeAg, or because of mutations in the core promoter, which affect transcription of the HBeAg coding region.

Sustained non-viraemia is prerequisite for a virological cure of hepatitis C virus (HCV) disease. We monitored serum HCV RNA in our patients during interferon therapy and For 6 months of follow-up. The rate of sustained responders (RNA-negative at the end of follow-up) differed significantly by whether or not RNA became sero-negative within 24 weeks of the initiation of therapy (71% vs 7%); and also by whether or not the RNA-seronegativity lasted for 12 weeks during therapy (88% vs 11%). Thus, by monitoring viraemia in individual patients, we can tailor the duration of therapy and minimize waste of interferon.

Nine (10%) out of 90 hepatitis C virus (HCV) isolates from hepatitis patients and commercial blood donors in Thailand were not classifiable into any of genotypes I/1a, II/1b, III/2a, IV/2b, V/3a or VI/3b by RT-PCR with type-specific primers deduced from the HCV core gene. These isolates were sequenced over a 1.6 kb stretch of the 5'-terminal sequence and 1.1 kb of the 3'-terminal sequence covering 30% of the entire genome. Based on two-by-two comparison and phylogenetic analyses of the nine Thailand isolates among themselves and with known full or partial sequences of previously reported HCV isolates, the Thailand isolates were classified into five genotypes not reported previously, viz. 6b, 7c, 7d, 9b and 9c. Along with HCV isolates reported already, they make at least nine major genetic groups of HCV which further break down into at least 28 genotypes with sequence similarity in the E1 gene (576 bp) of ≤ 80%. As many more HCV isolates of distinct genotypes are expected to be found throughout the world, it will become increasingly difficult to classify them by comparison of any partial sequences of the genome. Complete sequence data will be required for the full characterization and classification of HCV genotypes.

Hepatitis B virus (HBV) DNA clones were propagated from 57 carriers with antibody to hepatitis B e antigen (HBeAg) and sequenced within nucleotides (nt) 1685 to 1926 including the core promoter (nt 1732 to 1849) and the pre-C region (nt 1814 to 1900). Mutations in the core promoter or those in the pre-C region, or both, were detected in 328 (97.9%) of 335 clones from them. Five carriers were infected with HBV mutants with mutations in the core promoter alone, while 20 carriers were infected only with those in the pre-C region to abort the translation of HBeAg precursor; the remaining 32 carriers were infected with HBV mutants with mutations in both the core promoter and pre-C region. Some carriers infected with HBV with mutations in the core promoter exclusively had high HBV DNA titers, comparable with those in carriers infected with wild-type HBV, thereby indicating that such mutations would not affect the transcription of the HBV pregenome extensively. Two point mutations in the core promoter, from A to T at nt 1762 and From G to A at nt 1764, were most prevalent. The other mutations included a point mutation at either of the two nucleotides and their deletion. All of these mutations involved the TTAAA sequence (nt 1758 to 1762) at 28 hp upstream of the initiation site for shorter pre-C mRNAs (nt 1790 +/- 1). The ATAAATT sequence (nt 1789 to 1795) at 23 bp upstream of the initiation site for the pregenome RNA (nt 1818), however, remained intact in all 335 HBV DNA clones. HBV mutants with mutations in the core promoter, unaccompanied by pre-C mutations, prevailed and replaced wild-type HBV in two carriers as they seroconverted from HBeAg to the corresponding antibody. These results indicate that HBV mutants with an HBeAg- phenotype would be generated by mutations in the core promoter which might abort the transcription of pre-C mRNA but do not seriously affect that of pregenome RNA.

Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family, The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household.

Three chimpanzees persistently infected with hepatitis C virus of genotype II/1b were challenged with hepatitis C virus of genotype III/2a and 6 wk later with hepatitis C virus of genotype I/1a. They were tested for titers of total and genotype-specific hepatitis C virus RNA, as well as for serum transaminase levels, until 52 wk after the first challenge. One chimpanzee (CH489) with intermittent low hepatitis C virus RNA titers of genotype II/1b in serum was superinfected with hepatitis C virus of genotype III/2a between wk 1 and 7 after the challenge; superinfection was accompanied by fluctuating high transaminase levels. Later, the animal was superinfected with hepatitis C virus of genotype I/1a. Superinfection was accompanied by persistently high transaminase levels immediately after the challenge. Hepatitis C virus of genotype I/1a persisted, whereas hepatitis C virus of genotype II/1b was undetectable 22 wk after the challenge and thereafter. In another chimpanzee (CH353) with intermittent low hepatitis C virus RNA titers of genotype II/1b, hepatitis C virus of genotype III/2a induced fluctuating high levels of serum transaminases without revealing itself in serum. Then, HCV of genotype I/1a superinfected her, induced persistently high transaminase levels and took over HCV of genotype II/1b at 22 wk after the challenge and thereafter. The third chimpanzee (CH451) with persistently high HCV RNA titers of genotype II/1b did not reveal HCV RNA of genotype III/2a in serum after the challenge, although transaminases sharply increased. Low-titered HCV RNA of genotype I/1a was detected at 18 wk after the challenge. He continued to have elevated transaminase levels and high-titered HCV RNA of genotype II/1b throughout the observation period. These results indicate superinfection with HCV of different genotypes in chimpanzees, newly introduced HCV replacing or being expelled by the predecessor HCV with each infection inducing an episode of liver injury.

Objectives: Hepatitis C virus (HCV) infection induces extra-hepatic manifestations, most of which are considered to be mediated by circulating immune complexes. For evaluating this association in a wider perspective, complement activity was determined in sera from apparently healthy individuals, and hypocomplementemia was tested for correlation with HCV viremia. Methods: Sera from 10,532 voluntary blood donors were stored at 4 degrees C overnight, serially diluted 2-fold, and tested for hemolytic activity by a microtitration method and antibody to HCV (anti-HCV) by passive hemagglutination with recombinant HCV antigens of the second generation. HCV RNA was determined in sera with anti-HCV or hypocomplementemia, or both, by polymerase chain reaction with nested primers deduced from the 5'-noncoding region of the HCV genome. Results: Hypocomplementemia was detected in 53 (0.5%) of 10,532 donations and anti-HCV in 94 (0.9%). Anti-HCV was detected in 48 (91%) of the 53 sera with hypocomplementemia, more frequently than in 46 (0.44%) of 10,479 sera, without (p < 0.001). Among 94 sera positive for anti-HCV, HCV RNA was detected in 45 (94%) of 48 sera with hypocomplementemia, more often than in 10 (22%) of 46 sera without (p < 0.001). Conclusions: A close association of hypocomplementemia with HCV viremia among apparently healthy blood donors would reflect circulating immune complexes which may cause extrahepatic diseases, such as cryoglobulinemia and membranoproliferative glomerulonephritis, in some HCV carriers. The storage of sera from HCV carriers at 4 degrees C before the test would have contributed to a decreased hemolytic activity due to the cold activation of complement by cryoglobulins involving HCV.

Hepatitis C virus (HCV) RNA and genotypes, as well as markers of hepatitis B virus infection, were surveyed in 171 patients with chronic liver disease, 276 patients with chronic renal failure, and 961 blood donors in Thailand. HCV RNA was detected in 30 (23%) of 128 patients with nonalcoholic chronic liver disease and hepatitis B surface antigen (HBsAS) in 60 (47%), and both HCV RNA and HBsAS in 3; the cause of liver disease was not established in 41 (32%) patients. HCV RNA was detected in 44 (20%) of 221 patients on maintenance hemodialysis or with kidney transplantation, but in none of 55 patients on peritoneal dialysis. Antibodies to synthetic HCV core peptides were detected in 39 (4.1%) of sera from 961 blood donors, and HCV RNA was detected in 8 (0.8%). Of the 90 HCV RNA samples from patients and donors, genotype V prevailed (46%) followed by II (22%), I (14%), III (3%), and VI (2%); genotypes were not classifiable into any of I-VI in the remaining 10%. There were six sera which contained HCV RNA, but were without antibody to HCV detectable by the second-generation enzyme immunoassay. HCV RNA titers were high in four patients with kidney transplantation, but low in one patient with chronic liver disease and one patient on maintenance hemodialysis. HCV RNA at high titer (greater than or equal to 10(4)/ml) was not classifiable in one patient. These results indicate HCV of novel genotypes in Thailand, seronegative HCV infection in patients with kidney transplantation, and a low risk of HCV infection in patients treated by peritoneal dialysis. (C) 1994 Wiley-Liss, Inc.

A human plasma (inoculum one) containing hepatitis C virus (HCV) was passaged through eight chimpanzees in three generations. Of 10 HCV clones propagated from it, 7 were different in respect to the hypervariable region of E2/NS1 glycoprotein and they were named clones A, B, C, etc. A chimpanzee received inoculum one, and clone A accounted for 7 of the 10 clones from his acute-phase plasma (inoculum two). Five chimpanzees received inoculum two, and clone A accounted for 5 to 9 of the 10 clones each from their preacute plasma, which were pooled to make inoculum three. Of 10 HCV clones from inoculum three, 4 were A, 5 were B/B', and the remaining 1 was C. Two chimpanzees received inoculum three or its CsCl fraction, and all 40 clones from their acute-phase plasma were A. Thus, clone A in inoculum one was selected by chimpanzees during three passages. HCV virions in the three inocula were separated into free and immunoglobulin-bound forms by sucrose density fractionation. HCV virions in inocula one and two were heterogeneous in the sequence of hypervariable region and predominantly free of immunoglobulins. By contrast, inoculum three contained both free virions of predominantly A and immunoglobulin-bound virions which were heterogeneous. Antibodies to the hypervariable region were determined by enzyme immunoassays with overlapping synthetic decapeptides. Antibodies to clone A were detected in one chimpanzee who received inoculum two, and those to clones a and C in two chimpanzees including him and in inoculum three. Antibodies were not detectable in inoculum one or two or in the other chimpanzees. These results indicate that antibodies to the hypervariable region of E2/NS1 glycoprotein would be protective and contribute toward the selective replication of HCV in chimpanzees. (C) 1994 Academic Press, Inc.

Patients with chronic hepatitis C were treated with interferon (IFN) and followed for hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum. The response was correlated with decrease in serum levels of HCV RNA, as well as HCV genotypes and liver histopathology. Response to IFN, estimated by clearance of HCV RNA and normalization of aminotransferase levels at 6 months after the withdrawal of IFN, was observed in 11 (31%) of 35 patients infected with HCV of genotype II/1b, 13 (72%) of 18 with genotype III/2a, and 2 (33%) of 6 with genotype IV/2b; a single patient with genotype I/1a responded while the one doubly infected with HCV of genotypes II/1b and IV/2b did not. Response was seen in 10 (71%) of 14 patients with chronic persistent hepatitis, 14 (39%) of 36 with chronic active hepatitis 2A, and 3 (27%) of 11 with 2B. Response was achieved less often in patients with high than low pretreatment levels of HCV RNA. HCV RNA dropped sharply on a day after the start of IFN, and continued to decrease during the 2 weeks, irrespective of the response to IFN or HCV genotypes. In contrast, anti-HCV decreased more gradually and only in responders to IFN. These results support the rapid development of an IFN-mediated antiviral effect on HCV, and support therapeutic effects of IFN dependent on histopathology of liver as well as HCV RNA titers and genotypes,

Blood units from voluntary as well as commercial donors in Beijing, China, were tested for hepatitis C virus RNA and antibodies, and for serological markers of hepatitis B virus infection. HCV RNA was detected less frequently in 1909 voluntary donors (5 (0.3%)), than in 1017 commercial donors (58 (5.7%)) (p<0.001). Antibody to hepatitis C virus was detected by the second-generation enzyme immunoassay in 55 (87%) of 63 blood units with viremia. Evidence of present or past infection with hepatitis B virus was common both in voluntary (43.9%) and commercial (46.4%) donors. There were eight (13%) sera with HCV-RNA in which hepatitis C virus antibodies were not detectable by second-generation enzyme immunoassay. Of 63 HCV-RNA samples from donors, 33 (52%) were of genotype II, 18 (29%) of III and one (2%) of II+III. HCV-RNA in the remaining 11 (17%) were not classifiable into any of the genotypes I, II, III,;IV and V. Genotype II was more frequent in viremic donors with elevated alanine aminotransferase levels (13/18 or 72%) than in those with normal levels (20/45 or 44%). These results indicate a low prevalence of hepatitis C virus infection in the general population in Beijing, and the limitations of identifying sera with viremia by second-generation enzyme immunoassay. (C) Journal of Hepatology.

The entire nucleotide sequence was determined for eight hepatitis B virus (HBV) genomes from three symptom-free carriers, two patients with chronic persistent hepatitis and one patient with chronic active hepatitis, who were positive for antibody to hepatitis B e antigen (HBeAg). The two patients with chronic persistent hepatitis were tested again after they developed chronic active or fulminant hepatitis, making a total of eight samples. Six had a point mutation in the preC region prohibiting the encoding of HBeAg precursor, while the remaining two had a deletion of 8 or 21 nucleotides within the X gene upstream of the preC transcription initiation sites which would affect the X gene and the putative preC/C promoter. Most genomes from the three symptom-free carriers and the two patients with chronic persistent hepatitis, with HBV DNA levels of 10(2)-10(3)/ml, had deletion, frameshift mutation, initiation failure or a premature stop codon, rendering them replication-incompetent. In contrast, such mutations were rarely seen in HBV genomes from the two patients with chronic persistent hepatitis after they had developed active or fulminant hepatitis and from the patient with chronic active hepatitis, all of whom had vigorous HBV replication with serum HBV DNA from 10(6) to 10(9)/ml Unique mutations for amino acid changes were more frequent in HBV genomes with a higher replicative activity. These results indicate two kinds of HBV genomes with an HBeAg-minus phenotype, one with defects seriously affecting viral replication and the other without such defects, which would account for different clinical profiles in carriers with antibody to HBeAg. (C) 1994 Wiley-Liss, Inc.

Spouses of hepatitis C virus (HCV)-viremic chronic hepatitis patients were tested for antibodies to the core region-derived synthetic oligopeptides and for antibodies detectable by the second-generation enzyme immunoassay, and HCV RNA was determined in those who were positive for at least one of the above antibodies (anti-HCV). The prevalences of anti-HCV and HCV RNA were both significantly higher in 37 spouses of patients with a history of transfusion than in 55 spouses of patients with no such history: 49% vs. 16% for anti-HCV (P < 0.01) and 41% vs. 5% for HCV RNA (P < 0.05). Anti-HCV was detected in 11 (65%) of 17 spouses who had been married longer than 30 years since the patients received the transfusion, with an incidence significantly higher than in four (20%) of 20 spouses with exposure durations less than 30 years (P < 0.001), and HCV RNA was positive in 11 (65%) of the formers, the incidence being significantly greater than in four (20%) of the latter (P < 0.05). HCV-infected patients, in particular with a history of blood transfusion, are thus possible HCV transmission sources for their spouses, with the risk increasing with the duration of marriage, pointing to a need for follow-up not only for posttransfusion hepatitis C patients, but also for early diagnosis and treatment of their spouses.

A recombinant yeast-derived pre-S2 + S-containing hepatitis B vaccine (TGP-943) was clinically evaluated through three phases of testing in a total of 2137 volunteers. We observed the immunogenic purity of TGP-943 (phase 1), the inter-lot reproducibility of both safety and immunogenicity (phase 2), no significant side-effects, a high capability of inducing both anti-HBs and anti-pre-S2 antibodies (phases 1, 2 and 3), and an ability to induce seroconversion in the majority of vaccinees who had been non-responsive to conventional hepatitis B vaccines (phases 2 and 3). In conclusion, TGP-943 is a safe and tolerable vaccine, with special merits. the ability to induce an early anti-pre-S2 response that circumvents the problem of delayed appearance of anti-HBs, and efficacy in non-responders to previous vaccination.

Blood donors in two cities in Vietnam were tested for markers of hepatitis C virus (HCV) and hepatitis B virus infections. Antibody to HCV was detected by passive hemagglutination with antigens of the second generation in 101 (20.6%) of 491 donors in Ho Chi Minh City; it,vas detected less frequently (P < 0.001) in donors in Hanoi (4 [0.8%] of 499). HCV RNA was tested for in donors with antibody by PCR with nested primers from the 5'-noncoding region and detected in 79 donors in Ho Chi Minh City and 4 donors in Hanoi; HCV RNA was genotyped by PCR with type-specific primers from the core gene. Of 83 HCV carriers from Vietnam, 24 (29%) were infected with HCV of genotype I/1a, 19 (23%) were infected with II/1b, 4 (5%) were infected with III/2a, and 2 (2%) were infected with mixed genotypes (I/1a and II/1b); HCV genotypes in the remaining 34 (41%) donors, including all 4 donors in Hanoi, were not classifiable into I/1a, II/1b, III/2a, IV/2b, or V/3a. Of the 10 isolates with unclassifiable genotypes, 2 showed substantial sequence divergence within the S'-noncoding region from reported isolates with known genotypes (I/1a to 6a). An analysis of part of the core gene sequence indicated that six of the remaining isolates most likely represented new HCV genotypes. Hepatitis B surface antigen and the corresponding antibody, respectively, were detected in 15 (3.1% and 234 (47.7%) donors in Re Chi Minh City as well as in 15 (3.0%) and 248 (49.7%) donors in Hanoi. These results indicate an extensive spread of HCV among Ho Chi Minh City donors and HCV of novel genotypes in Vietnam.

Inhabitants and patients of two cities in Vietnam were tested for antibodies to hepatitis C virus (anti-HCV), hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs). Anti-HCV was detected in 43 (9%) of 491 individuals without liver disease in Ho Chi Minh, more frequently (P < 0.001) than in 18 (4%) of 511 in Hanoi. There was no apparent age-specific distribution of anti-HCV. Among inhabitants of both cities, HBsAg and anti-HBs were frequent, detected in 10-14% and 35-37%, respectively; the prevalence of anti-HBs increased in parallel with age. Among individuals at high risk, the prevalence of anti-HCV was particularly high in drug users (58/67 or 87%) and patients on maintenance haemodialysis (15/28 or 54%) or with haemophilia (7/24 or 29%) in Ho Chi Minh, and in drug users in Hanoi (61/200 or 31%). Prevalence of HBsAg and anti-HBs in high-risk groups was not different from those in the general population. Screening of anti-HCV in blood donors in Vietnam is of urgent necessity because blood supply is dependent on commercial blood donors, many of whom are drug users at high risk.

The entire nucleotide sequence of a hepatitis C virus (HCV) genome (NZL1) of genotype V/3a was determined from overlapping cDNA clones obtained from a human carrier in New Zealand. It comprised 9425 nucleotides (nt) including a 5'-untranslated region of 339 nt, a single large open reading frame encoding a polyprotein of 3021 amino acids, a 3'-untranslated region of 23 nt, and 3'-terminal poly(U) stretches of variable lengths. The NZL1 genome was compared with 15 HCV isolates of other genotypes for which the full-length sequence has been determined. It differed from them by 31.1 to 34.3% in nucleotide sequence identity and by 24.5 to 29.1% in amino acid sequence identity, confirming the distinction of genotype V/3a from the other isolates.

Type-specific antibodies were determined by enzyme-linked immunosorbent assay with antigen probes deduced from the core gene of hepatitis C virus (HCV) in 50 patients with chronic active hepatitis C who received interferon alfa. Type 1 antibody was detected in 16 (50%) of 32 patients infected with HCV of genotype II, and type 2 antibody in ten (56%) of 18 patients infected with HCV of genotype III or IV. Type 1 antibody was not detectable in any patients infected with HCV of genotype III or IV, while type 2 antibody was not detected in any patients infected with HCV of genotype II. Response to interferon occurred in six (38%) of 16 patients with type 1 antibody and eight (50%) of 16 without who were infected with HCV of genotype II. It was achieved in nine (90%) of ten patients with type 2 antibody and six (75%) of eight without antibody who were infected with HCV of genotype III or IV. The rate of response to interferon in patients infected with HCV of genotype III or IV (15/18 or 83%) was higher (P < 0.025) than that in patients infected with HCV of genotype II (14/32 or 44%). The response was seen much more frequently (P < 0.025) in patients with type 2 antibody (9/10 or 90%) than that in those with type 1 antibody (6/16 or 38%). These results indicate that determination of type-specific antibody to HCV would be useful for predicting response to interferon in patients with chronic hepatitis C.

Hepatitis B surface antigen (HBsAg) and hepatitis C virus (HCV) RNA were surveyed in patients in Yogyakarta, Indonesia, and their subtypes and genotypes were determined by serological methods and polymerase chain reaction with type-specific primers, respectively. Of 149 patients with chronic liver disease including 24 with chronic hepatitis, 86 with liver cirrhosis, and 39 with primary hepatocellular carcinoma, HBsAg was detected in 40 (27%) and HCV RNA in 48 (32%); one patient was positive both for HBsAg and HCV RNA. Thus, the cause of chronic liver disease was not identified in 62 (42%) patients. Of 58 patients on maintenance hemodialysis, four (7%) were positive for HBsAg and 44 (76%) for HCV RNA. Subtype adw was found in 34 (74%) of 46 HBsAg samples and adr in five (11%); compound subtypes, such as adyw and adyr were detected in the remaining seven (15%). Among HCV RNA samples from 48 patients with chronic liver disease, 23 (48%) were of genotype 11, 17 (35%) of genotype III and one (2%) of genotype V, in a distribution strikingly different from that of 44 samples from patients on maintenance hemodialysis, 39 (89%) of which were of genotype I and only one (2%) of genotype II. Genotypes were not classifiable in seven (15%) patients with liver disease and four (9%) patients on hemodialysis despite high HCV RNA titers in them all. These results indicate that different HCV genotypes prevail in patients with distinct diseases, as well as unclassifiable HCV genotypes in Indonesia. (C) 1994 Wiley-Liss, Inc.

Objective: Survey for markers of hepatitis C virus (HCV) infection in spouses of patients with HCV-related chronic liver disease. Design: Cross-sectional clinical, serologic, and molecular biological study of spouses of patients with HCV viremia and chronic liver disease. Setting: University and city hospitals. Participants: Spouses (52 men and 102 women; mean age, 56 +/- 11 years) of 154 patients with HCV viremia (102 men and 52 women; mean age, 58 +/- 10 years), of whom 66 had chronic hepatitis, 49 had liver cirrhosis, and 39 had primary hepatocellular carcinoma. Methods: Tests for HCV-associated antibodies were done using a second-generation enzyme immunoassay and immunoassays with synthetic oligopeptides deduced from the HCV core gene. Hepatitis C virus RNA was detected by polymerase chain reaction with primers deduced from the 5'-noncoding region and HCV genotypes by reaction with type-specific primers deduced from the HCV core gene. Results: Hepatitis C virus-associated antibodies were detected in 42 (27%) spouses, of whom 25 were also positive for HCV RNA. Of 112 (73%) spouses without detectable antibodies, 2 had chronic liver disease. The development of markers of HCV infection in spouses increased with the duration of marriage, ranging from 1 to 60 years (30 +/- 11 years). Conclusions: Spouses of patients with HCV viremia and chronic liver disease have an increased risk for acquiring HCV, which is proportional to the duration of marriage. They should be followed routinely for markers of HCV infection and liver disease.

Five isolates of hepatitis C virus (HCV) RNA from patients with chronic liver disease in Nepal were not classifiable into the known genotypes I/1a, II/1b, III/2a, IV/2b or V/3a using PCR with type-specific primers deduced from the HCV core gene. Their nucleotide sequences were determined for the 5'-terminal 1.5 kilobases and 3'-terminal 1.2 kilobases, covering 30% of the entire genome, and compared with each other and with reported sequences of HCV isolates of various genotypes. They were more similar to a reported HCV isolate (NZL1) of genotype V/3a (in 81.6 to 84.1% of their nucleotides and 85.7 to 88.7% of the deduced amino acid sequence) compared with the genotypes I/1a to IV/2b (in 69.3 to 74.7% and 72.3 to 77.4%, respectively). Hence they were considered to be variants of the third major group (group 3). The five HCV isolates shared 81.3 to 85.2% of nucleotide sequence and 85.4 to 89.3% of deduced amino acid sequence. Thus they were substantially different from each other. One of them was classified as genotype VI/3b due to an 88.2% similarity in nucleotide sequence to that of the reported HCV isolates of this genotype, whereas the remaining four were classified into provisional genotypes 3c, 3d, 3e and 3f. These HCV variants have evolved and remained in Nepal, and have not been observed in the other areas of the world.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.

A total of 145 patients with chronic liver disease, including 20 with chronic hepatitis, 63 with cirrhosis and 62 with primary hepatocellular carcinoma from Nepal were tested for markers of hepatitis B virus or hepatitis C virus infection. HBsAg was detected in 57 (39%) and hepatitis C virus RNA in 12 (8%); the cause of liver disease was not known in the remaining 76 (52%). HBsAg was found in 5 (1.3%) of 379 normal controls, whereas hepatitis C virus-associated antibodies were detected in 13 (3.4%), none of whom was positive for serum hepatitis C virus RNA. Subtypes of 102 HBsAg samples, from patients and asymptomatic carriers, were adw in 35 (34%), adr in 4 (4%) and ayw in 48 (47%); the remaining 15 (15%) were of atypical subtypes such as ad, ay and a. Of 12 hepatitis C virus RNA samples, genotype I was detected in 1, genotype II in 5 and genotype V in 1; the remaining five samples were not to be classified by polymerase chain reaction with primers specific for genotypes I to V deduced from hepatitis C virus core sequences, despite high hepatitis C virus RNA titers in all of them. Sequences of 192 amino acids in the entire El region of unclassifiable hepatitis C virus isolates from five patients differed from each other in 17% to 23%, and varied from reported isolates of defined genotypes in 13% to 44%. These results indicate that atypical subtypes of hepatitis B virus and novel genotypes of hepatitis C virus would prevail in Nepal.

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.