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Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease
Journal of Hepatology 1994; 21:468--473 Printed hi Denmark. All rights reserved Munksgaard. Copenhagen
Copyright © Journalof Hepatology 1994
Journal of Hepatology ISSN 0168-8278
Rapid Publication
Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease Eiji Mita, N o r i o Hayashi, Yoshiyuki K a n a z a w a , Hideki Hagiwara, Keiji Ueda, Akinori K a s a h a r a , Hideyuki F u s a m o t o and T a k e n o b u K a m a d a First Deparonent of Medicine, Osaka UniversiO, School of Medicine, Suita, Osaka, Japan
(Received 9 December 1993)
Hepatitis C virus genotype and the amounts of circulating HCV RNA are the most important factors in determining the efficacy of interferon therapy for chronic hepatitis C. To clarify the correlation of these two factors to the progression of liver disease, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured their HCV RNA titers (logarithmic transformed copy number/ml serum) by competitive reverse transcription-polymerase chain reaction. We found type II in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with hepatocellular carcinoma. Thus, there was no significant difference in the prevalence of type II among the various stages of chronic liver disease. We also found the RNA titer to be significantly higher in patients with chronic active hepatitis (8.0___0.8) than in those with chronic persistent hepatitis (7.0___1.0, p<0.001), and also those with cirrhosis (7.6___0.8, p<0.05) or hepatocellular carcinoma (7.7---0.8, p<0.05). When the titers were compared among genotypes, there was no significant difference between type II and III at any stage (type II vs. type III: chronic persistent hepatitis, 7.2___1.0 vs. 6.7_+0.8; chronic active hepatitis, 8.1--_0.7 vs. 7.8--- 1.0; cirrhosis, 7.7___0.8 vs. 7.8___0.7; hepatocellular carcinoma, 7.7___0.8 vs. 7.8___0.5). In conclusion, although genotype affects interferon therapy efficacy, it seems to have little influence on serum RNA levels and the progression of type C chronic liver disease. © Journal of Hepatology. Key words: Hepatitis C virus; HCV RNA titer; Hepatitis C virus genotype; Type C chronic liver disease
HCV is a positive-strand RNA virus and has one long open reading frame that consists of approximately 3000 amino acids. The partial similarity of this amino acid sequence to those of flaviviruses and pestiviruses suggests that they may correspond to NH2-core-envelope 1-envelope 2/non-structural protein (NS) 1-NS2-NS3-NS4-NS5COOH and be processed to each protein by host or viral proteases (1). Analysis of the nucleotide sequence and the deduced amino acid sequence indicated that many HCV cDNA isolates could be classified into several genotypes (2-5). The cDNA clones that belonged to the same genotype were more than 90% homologous to one another in the deduced amino acid sequences. Okamoto et al. (3) reported that most HCV cDNA clones isolated from Ja-
panese patients could be classified into four genotypes (type I-IV, according to the classification method they established). The assay for antibody to HCV has been established and has revealed that HCV is a major causative agent of non-A, non-B liver disease (6-8). Patients who are positive for anti-HCV usually have chronic infection, and some develop cirrhosis or hepatocellular carcinoma (HCC)(9). The most effective treatment for chronic hepatitis C is interferon (IFN) therapy, and type III and IV patients seem to respond better to it than type II patients (10,11). As another clinical measure, many investigators have tried to quantitate circulating HCV RNA. By competitive reverse transcription-polymerase chain reaction
Correspondence to: Norio Hayashi, M.D., First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka
565, Japan.
HCV GENOTYPE AND CHRONIC LIVER DISEASE (RT-PCR), we (12) demonstrated that HCV RNA titer was lower in patients with chronic persistent hepatitis than in those with chronic active hepatitis, cirrhosis or HCC. Lau et al. (13) showed that the serum RNA levels in patients with chronic persistent hepatitis were higher than in those with chronic active hepatitis or chronic active hepatitis plus cirrhosis by branched DNA signal amplification. However, few studies have dealt with the correlation of HCV genotype and R.NA titer to the progression of type C liver disease. In the present study, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured the amounts of HCV RNA in serum by competitive RT-PCR to clarify the correlation of these two factors to the progression of type C chronic liver disease.
Patients and Methods
Patients Of Japanese patients with type C chronic liver disease who were positive for second-generation assay of antiHCV, 148 patients (102 males, 46 females) were selected randomly for this study. All were positive for HCV RNA tested by RT-PCR amplifying 5'-noncoding region (5'NCR) (14-16). Patients who were positive for hepatitis B surface antigen (HBsAg) or had a high titer of antibody to hepatitis B core were excluded. The patients selected showed no evidence of alcoholic, autoimmune or druginduced hepatitis. They were categorized into four groups: chronic persistent hepatitis, chronic active hepatitis, cirrhosis and HCC. All the patients with chronic hepatitis, and 26 with cirrhosis were diagnosed by liver biopsy. The remaining 14 patients with cirrhosis were diagnosed by clinical observations including ultrasonic findings. All patients with HCC were diagnosed by angiography. Four patients with chronic persistent hepatitis, 43 with chronic active hepatitis, 11 with cirrhosis and 15 with HCC were tested in the previous study (12). Clinical and biochemical profiles of these patients are summarized in Table 1. Serum samples were stored at -80°C until use. Two liver pathology specialists checked the biopsy samples, when available, by microscopy for histological diagnosis. RNA extract and subtyping H C V Each serum sample (50/11) was mixed thoroughly with 400 /.tl of guanidinium thiocyanate solution (17), and RNA fractions were purified by repeated extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitation. These RNA fractions were pelleted with 10/.tg of
469 TABLE 1 Clinical and biochemicalcharacteristicsof 148 patients CPH Females/males Age ~[year] Transfusion history ALT~[IU/I]
CAH
LC
HCC
10/20 10/33 18/22 8/27 4 1 . 1 ± 8 . 6 50.6±9.3 59.1±8.1 64.5±7.1 9 13 10 11 88.7±108.8153.1±91.1 92.4±53.8 83.8±53.1
ALT, alanine aminotransferase; CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; LC, cirrhosis; HCC, hepatocellular carcinoma. "Data expressed as mean±S.D. tRNA (Sigma Chemical Co., St. Louis, MO) and resuspended in diethyl pyrocarbonate-treated TE buffer (10 mM of Tris-C1, pH 8.0; 1 mM of ethylenediaminetetraacetate, pH 8.0) for the following RT. The HCV genotype was determined by minor modifications of Okamoto's method (3,10). Briefly, the R.T and 40 cycles of PCR were performed using a set of genotype-universal outer primers. For 40 cycles of the following nested PCR, the genotype-universal sense primer and the mixture of genotype-specific anti-sense primers were used. When the nested PCR products were electrophoresed on a 3% agarose gel and observed under ultraviolet illumination, type I-specific DNA fragments were observed as a 57-bp band, whereas type II-, III- or IV-specific fragments were found as a 144-, 174- or 123-bp band, respectively. Quantitative competitive RT-PCR method HCV RNA molecules in circulation were quantified by the competitive RT-PCR method with amplification in 5'NCR (12,18). We previously isolated the HCV clone named M642 and prepared the mutated EcoRI site by site-directed mutagenesis. Using this mutated HCV clone, the competitive RNA templates that have a two-base mismatch to the target HCV RNA were transcribed in vitro. Increasing known copy numbers of these templates were added to the guanidinium solution at the step of RNA extraction from serum samples. RT and 40 cycles of PCR were carried out in the 5'-NCR. The PCR products were digested with EcoRI under the recommended condition for 2 h and separated on a 2% agarose gel. The amplified products derived from the mutated templates were digested with EcoRI and observed as two different bands (198 and 108 bp) after electrophoretic separation. However, the amplified products synthesized from serum samples were not digested and appeared as a discrete single band (306 bp). We therefore compared the signal for serum sample-derived products with the signal for competitive template-derived products and determined the competition equivalence points. The amounts of HCV RNA in serum were subjected to logarithm transformation as HCV RNA titer.
470
E. MITA et al.
Statistical analysis Statistical analysis for group comparisons was done by the chi-squared test or Fisher's exact test. The titers of HCV RNA were compared among groups by the MannWhitney U test.
Results
Prevalence of H C V genotype in various categories of type C chronic liver disease Among 148 patients with type C chronic liver disease, type II was observed in 116 (78.4%), whereas type III and IV were found in 16 (10.8%) and 7 (4.7%), respectively. Type I was not observed. In our study, double infection was found in five patients; three had type II and III and two had type II and IV. Four patients could not be classified. The prevalence of HCV genotypes in each category of type C chronic liver disease is summarized in Table 2. Type II was observed in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with HCC. We found no significant difference in the prevalence of type II among the various categories of type C chronic liver disease. On the other hand, type III was observed in three (10.0%) patients with chronic persistent hepatitis, five (11.6%) with chronic active hepatitis, five (12.5%) with cirrhosis and three (8.6%) with HCC. Type IV was found in two (6.7%) with chronic persistent hepatitis, two (4.7%) with chronic active hepatitis and three (7.5%) with cirrhosis. However, no patients with HCC were of type IV.
H C V RNA titer in various categories of type C chronic liver disease When the HCV R N A titers were compared among various categories of type C chronic liver disease (Fig. I), the titer of patients with chronic active hepatitis (8.0---0.8) was significantly higher than that of those with chronic persistent hepatitis (7.0___1.0) (p<0.001). It was also significantly higher than the titer of the patients with cirrhosis (7.6___0.8) (p<0.05) or with HCC (7.7___0.8) (p<0.05). Furthermore, the titer of those with cirrhosis or HCC was significantly higher than that of those with chronic persistent hepatitis (p<0.05, p<0.01). These significant differences were also observed for type II patients. The HCV RNA titer of the patients with chronic active hepatitis (8.1_+0.7) was significantly higher (p<0.001) than that of patients with chronic persistent hepatitis (7.2_+ 1.0). It was also significantly higher than patients with cirrhosis (7.7_+0.8, p<0.05) or HCC (7.7_+ 0.8, p<0.05). For type III patients, the titer of patients with chronic persistent hepatitis (6.7_+0.8) tended to be lower than other categories, but no difference in the titer was found among those with chronic active hepatitis (7.8__+1.0), cirrhosis (7.8_+0.7) and HCC (7.8_+0.5). When the titers were compared between type II and III, we found no difference at any stage of type C chronic liver disease. As the number of type IV patients was small, no definite conclusions could be reached, but the titer of these patients tended to be lower than that of type II or III patients.
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TABLE 2 Prevalence of each genotype in various categories of type C chronic liver disease Genotype
CPH
CAH
LC
HCC
II
23 (76.7%)
34 (79. I%)
29 (72.5%)
30 (85.7%)
III
3 (10.0%) 2 (6.7%)
5 (11.6%) 2 (4.7%)
5 (12.5%) 3 (7.5%)
3 (8.6%) 0 (0.0%)
Mixed
1 (3.3%)
2 (4.7%)
2 (5.0%)
0 (0.0%)
Unclassified
1 (3.3%)
0 (0.0%)
1 (2.5%)
2 (5.7%)
43
40
IV
Total
30
35
CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; LC, cirrhosis; HCC, hepatocellular carcinoma.
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~
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0%% •
•
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70
~
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•
•
oO0
S
CPH
CAH
LC
HCC
Fig. 1. Correlation between HCV genotype and RNA titer in various categories of type C chronic liver disease. Closed circles, type II; open triangles, type III; closed triangles, type IV. Patients with mixed type and unclassified were not included. Statistically significant differences were observed for the following: CPHtota I vs. CAHLo~ 07<0.001), LC,ol~t(p<0.05), HCCtot~t 07<0.01); C A H t o t a I vs. LC,ota t 07<0.05), HCCtot~l 07<0.05); CAHIt vs. CPH. (p<0.001), LC. (p<0.05), HCC, (p<0.05). (CPH=chronic persistent hepatitis; HCC=hepatocellular carcinoma; CAH=chronic active hepatitis; LC=cirrhosis).
HCV GENOTYPE AND CHRONIC LIVER DISEASE The HCV RNA titer of patients whose genotype was not classified was more than 6.0. We could detect the genotype of patients whose RNA titer was very low, such as 4.0 or 4.5 (data not shown). We suspect that the genotype of these patients could not be classified because the primers used were mismatched with their target sequences.
Discussion HCV genotype and HCV RNA titers were studied because of their importance in predicting the efficacy of I F N therapy (10,11). A better response to I F N therapy in type III or IV than in type II genotypes might indicate that type III or IV patients would show a better prognosis than type II patients. Contrary to expectations, the prevalence of each genotype in various stages of type C chronic liver disease did not differ among genotypes. The question then arises of why genotype-specific characteristics do not affect the progression of liver disease but do affect the efficacy of I F N therapy. The most acceptable explanation may be that when patients receive.a great amount of antiviral pressure, as in IFN therapy, HCV rapidly produces escape mutants to maintain chronic infection. In a previous report on differences in the efficacy of I F N therapy among genotypes (10), we suggested that type III-encoded R N A polymerase might not be as flexible as type II and that the mutation rate of the former might be lower. Thus HCV type III might be cleared by IFN therapy before producing escape mutants. Other anti-viral pressures may not be as strong as I F N therapy and thus may not lead to rapid mutation. We also studied the correlation of HCV RNA titer to the progression of type C chronic liver disease. HBsAg is thought to be affected by circulating HBV virions. Unfortunately, there is no assay system for HCV-associated antigens affected by circulating HCV particles because their amounts are not as great as HBV particles. Since the discovery of HCV (19), the detection of serum HCV R N A by RT-PCR has been used as a substitute assay of HCVassociated antigens (14,20,21). However, this method was limited to qualitative analysis. Thus investigators have attempted quantitation of serum HCV R N A by several methods (12,13,22). Generally speaking, the accuracy of these methods depends on the efficiency of amplification determined by the position of the primers and priming efficiency. Most important is the design of primers because HCV is known to be often mutated. We used a set of primers, BKP-7 and -8, for competitive RT-PCR, and detected serum HCV RNA in almost all patients with chronic HCV infection, with the PCR products observed as a discrete single band after electrophoretic separation.
471 Thus the position of both primers appeared to be correct. Next, we compared the sequence of the sense primer, BKP-7, with those of other HCV clones (HCV-l(23): type I; BK(24), J(25): type II; HC-J6(26): type III; HC-J7, J8(27): type IV) and found no difference in nucleic acid sequences between BKP-7 and these clones. We set the anti-sense primer, BKP-8, at position - 2 3 ~ + 2 . (Nucleotide numbering corresponds to that of the prototype HCV-I sequence (23).) Bukh et al. (28) compared the sequences of 81 HCV clones and demonstrated three perfectly conserved regions in 5'-NCR, one of them at position - 6 5 ~ - 3 . Furthermore, the sequence of C at position - 1 and the initiation codon of A T G at position + 1-+3 were perfectly conserved among these 81 HCV isolates. There was only one base mismatch at position - 2 or no mismatch between BKP-8 and these HCV clones. We used BKP-8 for RT, which was carried out at 37°C. This primer is thought to become tightly attached to target HCV RNA at this temperature because 21 bases at the Y-end of BKP-8 matched perfectly. Once the BKP-8 was used for first strand synthesis, the templates for PCR were found to have the same sequence as BKP-8. The amplification was done equally in the 5'-NCR among different genotypes. Needless to say, the ME642 that was used to produce the mutated RNA templates for competitive RTPCR had the same sequence as BKP-7 and -8, indicating that the efficiency of amplification was not determined by mutated variation among different genotypes. Many other factors may affect the accuracy of quantitation, such as the efficiency of RNA extraction and estimation of amplified products. The ratio of serum sample to RNA extraction buffer and the concentration of protein or lipid in serum may change the efficiency of RNA extraction. Thus, there are small differences in the serum HCV RNA levels among reports in the literature. In the present study, the HCV RNA titer of patients with chronic active hepatitis was significantly higher than in patients with chronic persistent hepatitis. A more active state of viral replication in the liver may induce higher amounts of circulating HCV in patients with chronic active hepatitis than in those with chronic persistent hepatitis. This tendency was also found by Kato et ai.(22). The results of the present study also demonstrated that the titer in chronic active hepatitis was significantly higher than in cirrhosis and HCC, but the mean value of the titer in chronic active hepatitis was only 0.3 or 0.4 higher than in cirrhosis or HCC, and 1.0 higher than in chronic persistent hepatitis. When liver specimens of type C patients with cirrhosis or HCC are observed microscopically, the severity of inflammation due to viral hepatitis varies among individuals. In some cases, inflammation was as severe as in patients with chronic active hepatitis, while
472
in other cases, it was as mild as in patients with chronic persistent hepatitis. These individual variations in the severity of liver inflammation a m o n g patients with cirrhosis or H C C may result in lower titers than in patients with chronic active hepatitis. Actually, in patients with cirrhosis or HCC, some had relatively low R N A titers such as 6.0 or 6.5. In contrast to our results, Kato et a1.(22) showed a slight increase in the a m o u n t s of HCV R N A in serum in the progression from chronic active hepatitis to cirrhosis, or cirrhosis plus HCC. The patients with cirrhosis or HCC in the present study may have included some with low-grade liver inflammation thus affecting the results. When the R N A titer was compared in each genotype among the different categories of type C chronic liver disease, the same tendency to increasing titers from chronic persistent hepatitis to cirrhosis/HCC to chronic active hepatitis was observed for type II. The increase in the R N A titers from chronic persistent hepatitis to chronic active hepatitis was also found in type III. Thus, viral replication seems to be most active in all genotypes in chronic active hepatitis. Next, the titers in each category were compared between type II and III genotypes. Type II titers were a little higher than type III in patients with chronic persistent hepatitis or chronic active hepatitis, but the mean difference was not significant. Furthermore, no difference was found in the HCV R N A titer between type II and III in patients with cirrhosis or HCC. According to these observations, we concluded that there was no significant difference in the serum HCV R N A levels between type II and III genotypes at any stage of chronic liver disease, and that genotype does not seem to determine the HCV R N A titer. In summary, although genotype affects the efficacy of I F N therapy, it seems to have little influence on the serum R N A levels and the progression of type C chronic liver disease.
Acknowledgements This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan, and the Research G r o u p into Intractable Hepatitis sponsored by the Ministry of Health and Welfare of Japan.
References 1. Houghton M, Weiner A, Han J, Kuo G, Choo QL. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology 1992; 14: 38 I-8. 2. Nakao T, Enomoto N, Takada N, Takada A, Date T. Typing
E. MITA et al. of hepatitis C virus genomes by restriction fragment length polymorphism. J Gen Virol 1991; 72: 2105-12. 3. Okamoto H, Sugiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73: 673-9. 4. Cha TA, Beall E, Irvine B, et al. At least five related, but distinct, hepatitis C viral genotypes exist. Proc Natl Acad Sci USA 1992: 89: 7144-8. 5. Stuyver L, Rossau R. Wyseur A, et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol 1993: 74: 1093-102. 6. Kuo G, Choo QL. Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989: 244: 362--4. 7. Yuki N, Hayashi N. Hagiwara H, et al. Serodiagnosis ofchronic hepatitis C in Japan by second-generation recombinant immunoblot assay. J Hepatol 1993: 17: 170-4. 8. Yuki N. Hayashi N, Hagiwara H, et al. HCV RNA and antibody to HCV core protein in Japanese patients with chronic liver disease. Dig Dis Sci 1992; 37: 1483-8. 9. Resnick RH, Stone K, Antonioli D. Primary hepatocellular carcinoma following non-A, non-B posttransfusion hepatitis. Dig Dis Sci 1983; 28:908-11. 10. Mita E. Hayashi N, Hagiwara H, et al. Predicting interferon therapy efficacy from hepatitis C virus genotype and RNA titer. Dig Dis Sci, in press. 1I. Takada N, Takase S, Enomoto N, Takada A, Date T. Clinical backgrounds of the patients having different types of hepatitis C virus genomes. J Hepatol 1992: 14: 35-40. 12. Hagiwara H, Hayashi N, Mita E, et al. Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease. Hepatology 1993; 17: 545-50. 13. Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-4. 14. Hagiwara H, Hayashi N, Mira E, et al. Detection of hepatitis C virus RNA in chronic non-A, non-B liver disease. Gastroenterology 1992; 102: 692-4. 15. Hagiwara H, Hayashi N, Mita E. et al. Detection of hepatitis C virus R.NA in serum of patients with chronic hepatitis C treated with interferon-ct. Hepatology 1992: 15: 37-41. 16. Takehara T, Hayashi N, Mita E, et al. Detection of the minus strand of hepatitis C virus RNA by reverse transcription and polymerase chain reaction: implications for hepatitis C virus replication in infected tissue. Hepatology 1992: 15: 387-90. 17. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1989: 7.18-7.22. 18. Hagiwara H, Hayashi N, Mita E, et al. Replicative state of hepatitis C virus as a predictive factor for sustained response to interferon therapy. Gastroenterology 1993; 104: 877-83. 19. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of cDNA clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 20. Weiner AJ, Kuo G, Bradley DW, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 1990: 335: I-3. 21. Garson JA, Tedder RS, Briggs M, et al. Detection of hepatitis C viral sequences in blood donations by "'nested" polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 22. Kato N, Yokosuka O, Hosoda K, Ito Y, Ohto M, Omata M. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: increase of the virus in advanced liver disease. Hepatology 1993; 18: 16-20. 23. Choo Q-L, Richman KH, Han JH, et al. Genetic organization
HCV GENOTYPE AND CHRONIC LIVER DISEASE and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991; 88: 2451-5. 24. Takamizawa A, Mori C, Fuke I, et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 1991; 65: 1105-13. 25. Kato N, Hijikata M, Ootsuyama Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 1990; 87: 9524-8. 26. Okamoto H, Okada S, Sugiyama Y, et al. Nucleotide sequence
473 of the genetic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. J Gen Virol 1991; 72: 2697-704. 27. Okamoto H, Kurai K, Okada S, et al. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 1992; 188: 331-341. 28. Bukh J, Purcell RH, Miller RH. Sequence analysis of 5' noncoding region of hepatitis C virus. Proc Natl Acad Sci USA 1992; 89: 4942-6.
Copyright © Journalof Hepatology 1994
Journal of Hepatology ISSN 0168-8278
Rapid Publication
Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease Eiji Mita, N o r i o Hayashi, Yoshiyuki K a n a z a w a , Hideki Hagiwara, Keiji Ueda, Akinori K a s a h a r a , Hideyuki F u s a m o t o and T a k e n o b u K a m a d a First Deparonent of Medicine, Osaka UniversiO, School of Medicine, Suita, Osaka, Japan
(Received 9 December 1993)
Hepatitis C virus genotype and the amounts of circulating HCV RNA are the most important factors in determining the efficacy of interferon therapy for chronic hepatitis C. To clarify the correlation of these two factors to the progression of liver disease, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured their HCV RNA titers (logarithmic transformed copy number/ml serum) by competitive reverse transcription-polymerase chain reaction. We found type II in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with hepatocellular carcinoma. Thus, there was no significant difference in the prevalence of type II among the various stages of chronic liver disease. We also found the RNA titer to be significantly higher in patients with chronic active hepatitis (8.0___0.8) than in those with chronic persistent hepatitis (7.0___1.0, p<0.001), and also those with cirrhosis (7.6___0.8, p<0.05) or hepatocellular carcinoma (7.7---0.8, p<0.05). When the titers were compared among genotypes, there was no significant difference between type II and III at any stage (type II vs. type III: chronic persistent hepatitis, 7.2___1.0 vs. 6.7_+0.8; chronic active hepatitis, 8.1--_0.7 vs. 7.8--- 1.0; cirrhosis, 7.7___0.8 vs. 7.8___0.7; hepatocellular carcinoma, 7.7___0.8 vs. 7.8___0.5). In conclusion, although genotype affects interferon therapy efficacy, it seems to have little influence on serum RNA levels and the progression of type C chronic liver disease. © Journal of Hepatology. Key words: Hepatitis C virus; HCV RNA titer; Hepatitis C virus genotype; Type C chronic liver disease
HCV is a positive-strand RNA virus and has one long open reading frame that consists of approximately 3000 amino acids. The partial similarity of this amino acid sequence to those of flaviviruses and pestiviruses suggests that they may correspond to NH2-core-envelope 1-envelope 2/non-structural protein (NS) 1-NS2-NS3-NS4-NS5COOH and be processed to each protein by host or viral proteases (1). Analysis of the nucleotide sequence and the deduced amino acid sequence indicated that many HCV cDNA isolates could be classified into several genotypes (2-5). The cDNA clones that belonged to the same genotype were more than 90% homologous to one another in the deduced amino acid sequences. Okamoto et al. (3) reported that most HCV cDNA clones isolated from Ja-
panese patients could be classified into four genotypes (type I-IV, according to the classification method they established). The assay for antibody to HCV has been established and has revealed that HCV is a major causative agent of non-A, non-B liver disease (6-8). Patients who are positive for anti-HCV usually have chronic infection, and some develop cirrhosis or hepatocellular carcinoma (HCC)(9). The most effective treatment for chronic hepatitis C is interferon (IFN) therapy, and type III and IV patients seem to respond better to it than type II patients (10,11). As another clinical measure, many investigators have tried to quantitate circulating HCV RNA. By competitive reverse transcription-polymerase chain reaction
Correspondence to: Norio Hayashi, M.D., First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka
565, Japan.
HCV GENOTYPE AND CHRONIC LIVER DISEASE (RT-PCR), we (12) demonstrated that HCV RNA titer was lower in patients with chronic persistent hepatitis than in those with chronic active hepatitis, cirrhosis or HCC. Lau et al. (13) showed that the serum RNA levels in patients with chronic persistent hepatitis were higher than in those with chronic active hepatitis or chronic active hepatitis plus cirrhosis by branched DNA signal amplification. However, few studies have dealt with the correlation of HCV genotype and R.NA titer to the progression of type C liver disease. In the present study, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured the amounts of HCV RNA in serum by competitive RT-PCR to clarify the correlation of these two factors to the progression of type C chronic liver disease.
Patients and Methods
Patients Of Japanese patients with type C chronic liver disease who were positive for second-generation assay of antiHCV, 148 patients (102 males, 46 females) were selected randomly for this study. All were positive for HCV RNA tested by RT-PCR amplifying 5'-noncoding region (5'NCR) (14-16). Patients who were positive for hepatitis B surface antigen (HBsAg) or had a high titer of antibody to hepatitis B core were excluded. The patients selected showed no evidence of alcoholic, autoimmune or druginduced hepatitis. They were categorized into four groups: chronic persistent hepatitis, chronic active hepatitis, cirrhosis and HCC. All the patients with chronic hepatitis, and 26 with cirrhosis were diagnosed by liver biopsy. The remaining 14 patients with cirrhosis were diagnosed by clinical observations including ultrasonic findings. All patients with HCC were diagnosed by angiography. Four patients with chronic persistent hepatitis, 43 with chronic active hepatitis, 11 with cirrhosis and 15 with HCC were tested in the previous study (12). Clinical and biochemical profiles of these patients are summarized in Table 1. Serum samples were stored at -80°C until use. Two liver pathology specialists checked the biopsy samples, when available, by microscopy for histological diagnosis. RNA extract and subtyping H C V Each serum sample (50/11) was mixed thoroughly with 400 /.tl of guanidinium thiocyanate solution (17), and RNA fractions were purified by repeated extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitation. These RNA fractions were pelleted with 10/.tg of
469 TABLE 1 Clinical and biochemicalcharacteristicsof 148 patients CPH Females/males Age ~[year] Transfusion history ALT~[IU/I]
CAH
LC
HCC
10/20 10/33 18/22 8/27 4 1 . 1 ± 8 . 6 50.6±9.3 59.1±8.1 64.5±7.1 9 13 10 11 88.7±108.8153.1±91.1 92.4±53.8 83.8±53.1
ALT, alanine aminotransferase; CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; LC, cirrhosis; HCC, hepatocellular carcinoma. "Data expressed as mean±S.D. tRNA (Sigma Chemical Co., St. Louis, MO) and resuspended in diethyl pyrocarbonate-treated TE buffer (10 mM of Tris-C1, pH 8.0; 1 mM of ethylenediaminetetraacetate, pH 8.0) for the following RT. The HCV genotype was determined by minor modifications of Okamoto's method (3,10). Briefly, the R.T and 40 cycles of PCR were performed using a set of genotype-universal outer primers. For 40 cycles of the following nested PCR, the genotype-universal sense primer and the mixture of genotype-specific anti-sense primers were used. When the nested PCR products were electrophoresed on a 3% agarose gel and observed under ultraviolet illumination, type I-specific DNA fragments were observed as a 57-bp band, whereas type II-, III- or IV-specific fragments were found as a 144-, 174- or 123-bp band, respectively. Quantitative competitive RT-PCR method HCV RNA molecules in circulation were quantified by the competitive RT-PCR method with amplification in 5'NCR (12,18). We previously isolated the HCV clone named M642 and prepared the mutated EcoRI site by site-directed mutagenesis. Using this mutated HCV clone, the competitive RNA templates that have a two-base mismatch to the target HCV RNA were transcribed in vitro. Increasing known copy numbers of these templates were added to the guanidinium solution at the step of RNA extraction from serum samples. RT and 40 cycles of PCR were carried out in the 5'-NCR. The PCR products were digested with EcoRI under the recommended condition for 2 h and separated on a 2% agarose gel. The amplified products derived from the mutated templates were digested with EcoRI and observed as two different bands (198 and 108 bp) after electrophoretic separation. However, the amplified products synthesized from serum samples were not digested and appeared as a discrete single band (306 bp). We therefore compared the signal for serum sample-derived products with the signal for competitive template-derived products and determined the competition equivalence points. The amounts of HCV RNA in serum were subjected to logarithm transformation as HCV RNA titer.
470
E. MITA et al.
Statistical analysis Statistical analysis for group comparisons was done by the chi-squared test or Fisher's exact test. The titers of HCV RNA were compared among groups by the MannWhitney U test.
Results
Prevalence of H C V genotype in various categories of type C chronic liver disease Among 148 patients with type C chronic liver disease, type II was observed in 116 (78.4%), whereas type III and IV were found in 16 (10.8%) and 7 (4.7%), respectively. Type I was not observed. In our study, double infection was found in five patients; three had type II and III and two had type II and IV. Four patients could not be classified. The prevalence of HCV genotypes in each category of type C chronic liver disease is summarized in Table 2. Type II was observed in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with HCC. We found no significant difference in the prevalence of type II among the various categories of type C chronic liver disease. On the other hand, type III was observed in three (10.0%) patients with chronic persistent hepatitis, five (11.6%) with chronic active hepatitis, five (12.5%) with cirrhosis and three (8.6%) with HCC. Type IV was found in two (6.7%) with chronic persistent hepatitis, two (4.7%) with chronic active hepatitis and three (7.5%) with cirrhosis. However, no patients with HCC were of type IV.
H C V RNA titer in various categories of type C chronic liver disease When the HCV R N A titers were compared among various categories of type C chronic liver disease (Fig. I), the titer of patients with chronic active hepatitis (8.0---0.8) was significantly higher than that of those with chronic persistent hepatitis (7.0___1.0) (p<0.001). It was also significantly higher than the titer of the patients with cirrhosis (7.6___0.8) (p<0.05) or with HCC (7.7___0.8) (p<0.05). Furthermore, the titer of those with cirrhosis or HCC was significantly higher than that of those with chronic persistent hepatitis (p<0.05, p<0.01). These significant differences were also observed for type II patients. The HCV RNA titer of the patients with chronic active hepatitis (8.1_+0.7) was significantly higher (p<0.001) than that of patients with chronic persistent hepatitis (7.2_+ 1.0). It was also significantly higher than patients with cirrhosis (7.7_+0.8, p<0.05) or HCC (7.7_+ 0.8, p<0.05). For type III patients, the titer of patients with chronic persistent hepatitis (6.7_+0.8) tended to be lower than other categories, but no difference in the titer was found among those with chronic active hepatitis (7.8__+1.0), cirrhosis (7.8_+0.7) and HCC (7.8_+0.5). When the titers were compared between type II and III, we found no difference at any stage of type C chronic liver disease. As the number of type IV patients was small, no definite conclusions could be reached, but the titer of these patients tended to be lower than that of type II or III patients.
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TABLE 2 Prevalence of each genotype in various categories of type C chronic liver disease Genotype
CPH
CAH
LC
HCC
II
23 (76.7%)
34 (79. I%)
29 (72.5%)
30 (85.7%)
III
3 (10.0%) 2 (6.7%)
5 (11.6%) 2 (4.7%)
5 (12.5%) 3 (7.5%)
3 (8.6%) 0 (0.0%)
Mixed
1 (3.3%)
2 (4.7%)
2 (5.0%)
0 (0.0%)
Unclassified
1 (3.3%)
0 (0.0%)
1 (2.5%)
2 (5.7%)
43
40
IV
Total
30
35
CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; LC, cirrhosis; HCC, hepatocellular carcinoma.
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LC
HCC
Fig. 1. Correlation between HCV genotype and RNA titer in various categories of type C chronic liver disease. Closed circles, type II; open triangles, type III; closed triangles, type IV. Patients with mixed type and unclassified were not included. Statistically significant differences were observed for the following: CPHtota I vs. CAHLo~ 07<0.001), LC,ol~t(p<0.05), HCCtot~t 07<0.01); C A H t o t a I vs. LC,ota t 07<0.05), HCCtot~l 07<0.05); CAHIt vs. CPH. (p<0.001), LC. (p<0.05), HCC, (p<0.05). (CPH=chronic persistent hepatitis; HCC=hepatocellular carcinoma; CAH=chronic active hepatitis; LC=cirrhosis).
HCV GENOTYPE AND CHRONIC LIVER DISEASE The HCV RNA titer of patients whose genotype was not classified was more than 6.0. We could detect the genotype of patients whose RNA titer was very low, such as 4.0 or 4.5 (data not shown). We suspect that the genotype of these patients could not be classified because the primers used were mismatched with their target sequences.
Discussion HCV genotype and HCV RNA titers were studied because of their importance in predicting the efficacy of I F N therapy (10,11). A better response to I F N therapy in type III or IV than in type II genotypes might indicate that type III or IV patients would show a better prognosis than type II patients. Contrary to expectations, the prevalence of each genotype in various stages of type C chronic liver disease did not differ among genotypes. The question then arises of why genotype-specific characteristics do not affect the progression of liver disease but do affect the efficacy of I F N therapy. The most acceptable explanation may be that when patients receive.a great amount of antiviral pressure, as in IFN therapy, HCV rapidly produces escape mutants to maintain chronic infection. In a previous report on differences in the efficacy of I F N therapy among genotypes (10), we suggested that type III-encoded R N A polymerase might not be as flexible as type II and that the mutation rate of the former might be lower. Thus HCV type III might be cleared by IFN therapy before producing escape mutants. Other anti-viral pressures may not be as strong as I F N therapy and thus may not lead to rapid mutation. We also studied the correlation of HCV RNA titer to the progression of type C chronic liver disease. HBsAg is thought to be affected by circulating HBV virions. Unfortunately, there is no assay system for HCV-associated antigens affected by circulating HCV particles because their amounts are not as great as HBV particles. Since the discovery of HCV (19), the detection of serum HCV R N A by RT-PCR has been used as a substitute assay of HCVassociated antigens (14,20,21). However, this method was limited to qualitative analysis. Thus investigators have attempted quantitation of serum HCV R N A by several methods (12,13,22). Generally speaking, the accuracy of these methods depends on the efficiency of amplification determined by the position of the primers and priming efficiency. Most important is the design of primers because HCV is known to be often mutated. We used a set of primers, BKP-7 and -8, for competitive RT-PCR, and detected serum HCV RNA in almost all patients with chronic HCV infection, with the PCR products observed as a discrete single band after electrophoretic separation.
471 Thus the position of both primers appeared to be correct. Next, we compared the sequence of the sense primer, BKP-7, with those of other HCV clones (HCV-l(23): type I; BK(24), J(25): type II; HC-J6(26): type III; HC-J7, J8(27): type IV) and found no difference in nucleic acid sequences between BKP-7 and these clones. We set the anti-sense primer, BKP-8, at position - 2 3 ~ + 2 . (Nucleotide numbering corresponds to that of the prototype HCV-I sequence (23).) Bukh et al. (28) compared the sequences of 81 HCV clones and demonstrated three perfectly conserved regions in 5'-NCR, one of them at position - 6 5 ~ - 3 . Furthermore, the sequence of C at position - 1 and the initiation codon of A T G at position + 1-+3 were perfectly conserved among these 81 HCV isolates. There was only one base mismatch at position - 2 or no mismatch between BKP-8 and these HCV clones. We used BKP-8 for RT, which was carried out at 37°C. This primer is thought to become tightly attached to target HCV RNA at this temperature because 21 bases at the Y-end of BKP-8 matched perfectly. Once the BKP-8 was used for first strand synthesis, the templates for PCR were found to have the same sequence as BKP-8. The amplification was done equally in the 5'-NCR among different genotypes. Needless to say, the ME642 that was used to produce the mutated RNA templates for competitive RTPCR had the same sequence as BKP-7 and -8, indicating that the efficiency of amplification was not determined by mutated variation among different genotypes. Many other factors may affect the accuracy of quantitation, such as the efficiency of RNA extraction and estimation of amplified products. The ratio of serum sample to RNA extraction buffer and the concentration of protein or lipid in serum may change the efficiency of RNA extraction. Thus, there are small differences in the serum HCV RNA levels among reports in the literature. In the present study, the HCV RNA titer of patients with chronic active hepatitis was significantly higher than in patients with chronic persistent hepatitis. A more active state of viral replication in the liver may induce higher amounts of circulating HCV in patients with chronic active hepatitis than in those with chronic persistent hepatitis. This tendency was also found by Kato et ai.(22). The results of the present study also demonstrated that the titer in chronic active hepatitis was significantly higher than in cirrhosis and HCC, but the mean value of the titer in chronic active hepatitis was only 0.3 or 0.4 higher than in cirrhosis or HCC, and 1.0 higher than in chronic persistent hepatitis. When liver specimens of type C patients with cirrhosis or HCC are observed microscopically, the severity of inflammation due to viral hepatitis varies among individuals. In some cases, inflammation was as severe as in patients with chronic active hepatitis, while
472
in other cases, it was as mild as in patients with chronic persistent hepatitis. These individual variations in the severity of liver inflammation a m o n g patients with cirrhosis or H C C may result in lower titers than in patients with chronic active hepatitis. Actually, in patients with cirrhosis or HCC, some had relatively low R N A titers such as 6.0 or 6.5. In contrast to our results, Kato et a1.(22) showed a slight increase in the a m o u n t s of HCV R N A in serum in the progression from chronic active hepatitis to cirrhosis, or cirrhosis plus HCC. The patients with cirrhosis or HCC in the present study may have included some with low-grade liver inflammation thus affecting the results. When the R N A titer was compared in each genotype among the different categories of type C chronic liver disease, the same tendency to increasing titers from chronic persistent hepatitis to cirrhosis/HCC to chronic active hepatitis was observed for type II. The increase in the R N A titers from chronic persistent hepatitis to chronic active hepatitis was also found in type III. Thus, viral replication seems to be most active in all genotypes in chronic active hepatitis. Next, the titers in each category were compared between type II and III genotypes. Type II titers were a little higher than type III in patients with chronic persistent hepatitis or chronic active hepatitis, but the mean difference was not significant. Furthermore, no difference was found in the HCV R N A titer between type II and III in patients with cirrhosis or HCC. According to these observations, we concluded that there was no significant difference in the serum HCV R N A levels between type II and III genotypes at any stage of chronic liver disease, and that genotype does not seem to determine the HCV R N A titer. In summary, although genotype affects the efficacy of I F N therapy, it seems to have little influence on the serum R N A levels and the progression of type C chronic liver disease.
Acknowledgements This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan, and the Research G r o u p into Intractable Hepatitis sponsored by the Ministry of Health and Welfare of Japan.
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