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Hepatitis C virus genotypes and quasispecies
Hepatitis C Virus Genotypes and Quasispecies Gary L. Davis, MD
Genetic heterogeneity is a hallmark of the hepatitis C virus, as a result largely of the infidelity of viral RNA– dependent RNA polymerase. Random nucleotide substitutions are introduced at a very high rate. The existence of genotypes was confirmed by statistical and mathematical techniques, and the relation of the genotypes to each other has been determined. There are six major genotypes, each with multiple subtypes. Isolates of the same genotype have an average sequence homology of 95%, but different genotypes have sequence similarity of approximately 65% on average. The nucleotide sequence in portions of the hepatitis C viral genome, including the 5ⴕ noncoding region, part of the core gene, and other nonstructural proteins, is highly conserved. Genotype analysis typically utilizes these highly conserved regions. There are many techniques for determining viral genotype, and in general, concordance between techniques is good. Methods most commonly used for assigning hepatitis C virus (HCV) genotypes in clinical practice include restriction fragment length polymorphism analysis and the reverse hybridization line probe assay (LiPA; Innogenetics, Ghent, Belgium). The worldwide distribution of HCV genotypes has been determined; some genotypes are highly characteristic of certain areas. The most common subtypes, 1 and 2, are less genetically diverse than the others and are more widely distributed. The impact of genotype on disease course is controversial, but recent data suggest that there is a genotypedependent differential response to therapy. Quasispecies refers to evolution of a highly related but genetically heterogeneous population of HCV isolates. The pathobiological and clinical implications of HCV quasispecies are poorly understood. Am J Med. 1999;107(6B):21S–26S. © 1999 by Excerpta Medica, Inc.
From the Department of Medicine, Section of Hepatobiliary Diseases, University of Florida, Gainesville, Florida. Requests for reprints should be addressed to Gary L. Davis, MD, Department of Medicine, Section of Hepatobiliary Diseases, University of Florida, 1600 South West Archer Road, PO Box 100214, Gainesville, Florida 32610 – 0214. © 1999 by Excerpta Medica, Inc. All rights reserved.
T
here are a variety of ways to categorize disease and the agents that cause them. Phenotype distinctions are based on the clinical presentation and behavior of a disease. Antibody neutralization of a specific antigen defines a serotype; this is typically used in distinguishing bacterial strains. Genotype and quasispecies refer to relatedness on a molecular or genetic level. Differences between genotypes are relatively large—there are nucleotide differences in 30% of the viral sequence. Quasispecies represent minor molecular variations, with only 1% or 2% nucleotide heterogeneity. The striking genetic heterogeneity of hepatitis C virus (HCV) is well recognized. This genetic diversity includes major genotypes (six), with numerous subtypes (over 80) and minor variants called “quasispecies.” The clinical implications of these variants, techniques for their identification, and their geographic distribution will be described.
MECHANISM OF GENOTYPE EVOLUTION During replication of the hepatitis C virus, RNA-dependent RNA polymerase frequently introduces random nucleotide errors. This results in a relatively high rate of spontaneous nucleotide substitutions, between 10⫺2 and 10⫺3/site/year. These replication errors yield 10 to 100 viable replacements per site per year. Whether a given nucleotide substitution results in a viable mutant is site dependent; different regions of the hepatitis C viral genome vary in their level of genetic conservation. Highly conserved areas include those coding for crucial functions, such as polymerase and other nonstructural proteins, core protein, the 5⬘ noncoding region, and the internal ribosome entry site. Nucleotide substitutions in these regions are less likely to be viable, because changes may result in altered structure that precludes function. Other regions of the genome are more tolerant of nucleotide substitutions, including the hypervariable regions. Such areas would be optimal for identifying subtle sequence variations, as in quasispecies analyses. The high rate of random nucleotide substitutions introduced by the viral RNA polymerase and the constant introduction of replication errors results in slow genetic evolution. As a consequence, HCV is a highly heterogeneous virus, with only approximately 70% homology between all isolates. The basis for classification into genotypes is genetic, or molecular, relatedness. Genotypes represent the molecular distance between different isolates of HCV. Because comparison of full0002-9343/99/$20.00 21S PII S002-9343(99)00376-9
A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis
Figure 1. Phylogenetic tree showing relatedness of six major hepatitis C genotypes and subtypes; produced by analysis of NS-5 sequences from 76 isolates of HCV. (Adapted with permission from Hepatology.1)
length viral sequences is impractical, several approaches have been developed to determine genetic relatedness of HCV isolates. Initially, investigators used direct sequencing of only the hypervariable region. Later, at least two regions were sequenced. Another method is phylogenetic tree analysis, which uses a variety of mathematical and statistical models to define genotype differences based on predicted changes within nucleotides. The more changes predicted, the greater the molecular distance between isolates. In 1993, Simmonds et al1 from Scotland published the phylogenetic tree analysis of the genotypes of HCV shown in Figure 1. It schematically shows the genetic distance between isolates. There are six major genotypes of HCV and at least 80 subtypes. Other genotypes have been defined, but subsequent evaluation has revealed that genotypes 7, 8, and 9 are actually subtypes of genotype 6.2 Further study of proposed genotype 10 showed that that is a subtype of genotype 3B. At this point, it is believed that there are six major genotypes of the HCV.
methods that we will be using clinically and are most familiar to us are the PCR-based approaches, which include sequencing, type specific primers, selective hybridization, which is the basis of the LiPA assay, and restriction fragment length polymorphism (RFLP). The LiPA test is based on differences coded by the NS4 region, and it detects genotypes 1 through 6. Overall, concordance between most of the different genotyping methods exceeds 93%.3 One exception is core-region genotype specific primers, the initial method described in Japan by Okamoto and colleagues.3a That technique is not as sensitive as others for the identification of genotype 1B. Both the RFLP4 and the LiPA technique that target the most highly conserved area of the HCV genome, the 5⬘ noncoding region, are sensitive, convenient, and economical. In addition, RFLP and LiPA results show high concordance with the more laborious sequencing and primerspecific PCR methods.3
HCV GENOTYPING ASSAYS
GEOGRAPHIC DISTRIBUTION OF HCV GENOTYPES
There are a variety of techniques for determining the genotype of an HCV isolate, which basically fall into two categories: PCR-based approaches and genotype-specific antibody approaches. These techniques identify different conserved portions of the HCV genome (Figure 1). The
Throughout the world, HCV genotypes differ with geographic region, as shown in Figure 2. HCV genotype 1 accounts for two thirds of HCV-infected individuals in the United States, with approximately equal numbers infected with genotypes 1A and 1B. Another 10% are in-
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A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis
Figure 2. Worldwide distribution of predominant hepatitis C genotypes.
fected with genotype 2, and 6% are infected with genotype 3. Approximately 10% are infected with mixed genotypes, and the genotype cannot be determined in the remainder.5 As in the United States, genotype 1 (specificially 1B) is predominant in Europe. Genotypes 2 and 3 are typically found in 15% to 30% of infected Europeans. Genotype 3 is very common in some parts of Europe; in Scandinavia, for example, 50% of HCV infections are genotype 3. Genotype 3 is also prevalent in the Far East (especially in Thailand), Pakistan, parts of India, and Australia. Genotype 4 is found almost exclusively in the Middle East. Genotype 5 is nearly always found in South Africa, and genotype 6 largely in Hong Kong.6,7
alcohol use, and response to therapy, were ignored.5,8 –11 Similarly, the impact of genotype on clinical and histologic behavior after liver transplantation has been extensively studied, with no definitive answers. Overall, there has been a suggestion in the literature that infection with genotype 1B is associated with more aggressive histology and greater recurrence after transplantation. Terrault et al12 analyzed HCV genotypes in clinic patients with liver disease and hepatic failure, and those that received liver transplants. Table 1 shows that similar proportions of patients with genotype 1 are seen in clinic and receive transplants, but proportionately more patients with genotype 2 and 3 receive liver transplants than are seen in clinic. These results suggest that genotype 1 is not associ-
IMPACT OF HCV GENOTYPE ON NATURAL HISTORY AND TREATMENT
Table 1. Impact of Hepatitis C Virus Genotype on Natural History
Whether HCV genotype affects the natural history and clinical behavior of hepatitis C has been debated in the literature from the time that genotypic distinctions were first noted. Many studies have focused on whether infection with genotype 1B leads to worse histology, but no consensus has been reached. In many studies, genotype was the only variable considered, and important other variables, including duration of infection, concomitant
Patients (%)* Genotype 1 2/3
Seen in Clinics
Received Transplant
67 16
61 25
* Patients at the University of California–San Francisco Transplant Center. Adapted from J Med Virol.12
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Figure 3. Response to therapy with ribavirin and interferon alfa-2b (Rebetron) or interferon alfa-2b alone (Intron A) in previously untreated patients according to hepatitis C genotype; m ⫽ months. (Adapted with permission from N Engl J Med.22)
ated with a worse prognosis.13–21 It is clear that more rigorous study is necessary. Genotype differences may be of considerable clinical importance if they are shown to reflect differences in response to therapy. McHutchison et al22 examined the impact of HCV genotype on response to combination therapy with ribavirin and interferon alfa-2b and interferon alone. Figure 3 shows the marked difference in response to therapy in treatment-naı¨ve patients infected with genotype 1 as compared with genotypes 2 and 3. Nearly 70% of patients with genotype 2 or 3 experienced sustained responses to combination therapy, even with the shorter duration of treatment (6 months). In comparison, 12 months of combination therapy led to a sustained response in only 30% of patients infected with genotype 1. Similar results were noted in patients who failed prior interferon therapy. Additionally, 6 months of combination therapy with ribavirin and interferon alfa-2b led to sustained responses in 75% of genotype 2 or 3 relapsers, but only in 30% of relapsers infected with HCV genotype 1.22 These results suggest that genotype influences response to therapy and indicate a need for development of aggressive therapies specifically directed toward resistant HCV genotype 1. Because no specific therapies for HCV genotype 1 exist and some people with genotype 1 do respond to therapy, genotyping to determine eligibility for treatment is not recommended.
QUASISPECIES The term “quasispecies” refers to pools of related genetic variants with only minor sequence differences (i.e., less than 5% of the viral genome). Although the differential 24S December 27, 1999 THE AMERICAN JOURNAL OF MEDICINE威
response to therapy according to HCV genotype indicates that genotype differences are clinically significant, the evidence to date is far less compelling for clinical implications of quasispecies differences. Thus, testing for quasispecies in an individual is not yet feasible in clinical practice. There are a few points regarding quasispecies that are worth mentioning, however. In a given inoculum, there may be a large number of quasispecies, but not all quasispecies are infectious. There is a range of replicative efficiency among quasispecies.23 The diversity of quasispecies in any individual is related to the size of the original inoculum.24 However, the evolution of quasispecies from a common inoculum varies with the host. Studies of HCV transmission from mother to child have demonstrated that the quasispecies that becomes most numerous in the child may not have been the most common one in the mother at the time of viral transmission. Host-mediated reasons for these differences may be related to humoral or cellular immune responses to the distinct viral variants. Quasispecies may differ in liver and plasma, as well.25 Future studies may demonstrate the clinical significance of this difference. Limited studies have indicated that quasispecies differ with respect to the ALT elevations they induce, and some data suggest that greater diversity in HCV quasispecies is correlated with more severe liver disease.26 –28 This is thought to be a result of an increased number of targets for cytotoxic T-lymphocyte attack in the liver, but this mechanism has not been confirmed.26,27 Quasispecies also appear to differ in their response to interferon-alfa therapy.2,24 Interferon therapy reduces heterogeneity as viral replication decreases.28 However, Volume 107 (6B)
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characterization studies have shown that after interferon therapy, different HCV quasispecies may become dominant. Finally, it appears that patients with more than two major quasispecies may respond poorly to interferon therapy.28,29 The quasispecies profile also often changes after liver transplantation. This implies a role for the liver, as well as host immune responses, in selection of predominant quasispecies.30,31
SUMMARY Hepatitis C virus is a heterogeneous virus with a high mutation rate. Major variants with up to 30% sequence difference represent the six major genotypes; multiple subtypes exist within each genotype. Minor sequence variations, on the order of 1% to 2%, are common; these quasispecies exist as a highly related but heterogeneous pool of isolates. Genotypes clearly show differences in their geographic distribution, with type 1B most common in the United States. The impact of genotype on the natural history of hepatitis C virus infection is controversial, but recent data show that genotype impacts significantly on response to therapy. This may ultimately influence choice and duration of therapy and perhaps affect patient selection for treatment, but such recommendations do not yet exist.
REFERENCES 1. Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes. [Letter.] Hepatology. 1994;19:1321–1324. 2. Mizokami M, Lau JYN, Suzuki K, et al. Differential sensitivity of hepatitis C virus quasispecies to interferon-␣ therapy. J Hepatol. 1994;21:884 – 886. 3. Lau JYN, Mizokami M, Kolberg JA, et al. Application of six hepatitis C virus subtyping systems to sera of patients with chronic hepatitis C in the United States. J Infect Dis. 1994; 171:281–289. 3a. 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– 679. 4. Davidson F, Simmonds P, Ferguson JP, et al. Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5⬘ noncoding region. J Gen Virol. 1995;76:1197–1204. 5. Lau JY, Davis GL, Prescott LE, et al. Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Hepatitis Interventional Therapy Group. Ann Intern Med. 1996;124:868 – 876. 6. McOmish F, Yap PL, Dow BC, et al. Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative survey. J Clin Microbiol. 1994;32: 884 – 892. 7. Mellor J, Holmes EC, Jarvis LM, et al. Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. J Gen Virol. 1995;76:2493–2507.
8. Mihm S, Fayyazi A, Hartmann H, et al. Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype. Hepatology. 1997;25:735–739. 9. Nousbaum JB, Pol S, Nalpas B, et al. Hepatitis C virus type 1b (II) infection in France and Italy. Collaborative Study Group. Ann Intern Med. 1995;1222:161–168. 10. Silini E, Bono F, Cividini A, et al. Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities. Hepatology. 1995;21:285–290. 11. Zeuzem S, Franke A, Lee JH, et al. Phylogenetic analysis of hepatitis C virus isolates and their correlation to viremia, liver function tests, and histology. Hepatology. 1996;24: 1003–1009. 12. Terrault NA, Dailey PJ, Ferrell L, et al. Hepatitis C virus: quantitation and distribution in liver. J Med Virol. 1997;51: 217–224. 13. Zhou S, Terrault NA, Ferrell L, et al. Severity of liver disease in liver transplantation recipients with hepatitis C virus infection: relationship to genotype and level of viremia. Hepatology. 1996;24:1041–1046. 14. Feray C, Gigou M, Samuel D, et al. Influence of the genotypes of hepatitis C virus on the severity of recurrent liver disease after liver transplantation. Gastroenterology. 1995; 108:1088 –1096. 15. Gane EJ, Portmann BC, Naoumov NV, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med. 1996;334:815– 820. 16. Pageaux GP, Ducos J, Mondain AM, et al. Hepatitis C virus genotypes and quantitation of serum hepatitis C virus RNA in liver transplant recipients: relationship with severity of histological recurrence and implications in the pathogenesis of HCV infection. Liver Transpl Surg. 1997; 3:501–505. 17. Belli LS, Silini E, Alberti A, et al. Hepatitis C virus genotypes, hepatitis, and hepatitis C virus recurrence after liver transplantation. Liver Transpl Surg. 1996;2:200 –205. 18. Charlton M, Seaberg E, Wiesner R, et al. Predictors of patients and graft survival following liver transplantation for hepatitis C. Hepatology. 1998;28:823– 830. 19. Berg T, Hopf U, Bechstein WO, et al. Pretransplant virological markers hepatitis C virus genotype and viremia level are not helpful in predicting individual outcomes after orthotopic liver transplantation. Transplantation. 1998;66:225– 228. 20. Vargas HE, Wang LF, Laskus T, et al. Distribution of infecting hepatitis C virus genotypes in end stage liver disease patients at a large American transplantation center. J Infect Dis. 1997;175:448 – 450. 21. Gayowski T, Singh N, Marino IR, et al. Hepatitis C virus genotypes in liver transplant recipients: impact on posttransplant recurrence, infections, response to interferonalpha therapy and outcome. Transplantation. 1997;64:422– 426. 22. McHutchison JG, Gordon SG, Schiff ER, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med. 1998;339:1485– 1492. 23. Sugitani M, Shikata T. Comparison of amino acid sequences in hypervariable region-1 of hepatitis C virus clones between human inocula and the infected chimpanzee sera. Virus Res. 1998;56;177–182. 24. Pawlotsky JM, Pellerin M, Bouvier M, et al. Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infec-
December 27, 1999
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A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis tion and responses to interferon alfa therapy in patients with chronic hepatitis C. J Med Virol. 1998;54:256 –264. 25. Maggi F, Fornai C, Vatteroni ML, et al. Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma. J Gen Virol. 1997;78:1521–1525. 26. Yuki N, Hayashi N, Moribe T, et al. Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies. Hepatology. 1997;25: 439 – 444. 27. Hayashi J, Kishihara Y, Yamaji K, et al. Hepatitis C viral quasispecies and liver damage in patients with chronic hepatitis C virus infection. Hepatology. 1997;25:697–701.
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28. Gonzalez-Peralta RP, Qian K, She YS, et al. Clinical implications of viral quasispecies in chronic hepatitis C. J Med Virol. 1996;49:242–247. 29. Toyoda H, Kumada T, Nakano S, et al. Quasispecies nature of hepatitis C virus and response to alpha interferon: significance as a predictor of direct response to interferon. J Hepatol. 1997;26:6 –13. 30. Yun Z, Barkholt L, Sonnerborg A. Dynamic analysis of hepatitis C virus polymorphism in patients with orthotopic liver transplantation. Transplantation. 1997;64:170 –172. 31. Lawal Z, Petrik J, Wong VS, et al. Hepatitis C virus genomic variation in untreated and immunosuppressed patients. Virology. 1997;228:107–111.
Volume 107 (6B)
Genetic heterogeneity is a hallmark of the hepatitis C virus, as a result largely of the infidelity of viral RNA– dependent RNA polymerase. Random nucleotide substitutions are introduced at a very high rate. The existence of genotypes was confirmed by statistical and mathematical techniques, and the relation of the genotypes to each other has been determined. There are six major genotypes, each with multiple subtypes. Isolates of the same genotype have an average sequence homology of 95%, but different genotypes have sequence similarity of approximately 65% on average. The nucleotide sequence in portions of the hepatitis C viral genome, including the 5ⴕ noncoding region, part of the core gene, and other nonstructural proteins, is highly conserved. Genotype analysis typically utilizes these highly conserved regions. There are many techniques for determining viral genotype, and in general, concordance between techniques is good. Methods most commonly used for assigning hepatitis C virus (HCV) genotypes in clinical practice include restriction fragment length polymorphism analysis and the reverse hybridization line probe assay (LiPA; Innogenetics, Ghent, Belgium). The worldwide distribution of HCV genotypes has been determined; some genotypes are highly characteristic of certain areas. The most common subtypes, 1 and 2, are less genetically diverse than the others and are more widely distributed. The impact of genotype on disease course is controversial, but recent data suggest that there is a genotypedependent differential response to therapy. Quasispecies refers to evolution of a highly related but genetically heterogeneous population of HCV isolates. The pathobiological and clinical implications of HCV quasispecies are poorly understood. Am J Med. 1999;107(6B):21S–26S. © 1999 by Excerpta Medica, Inc.
From the Department of Medicine, Section of Hepatobiliary Diseases, University of Florida, Gainesville, Florida. Requests for reprints should be addressed to Gary L. Davis, MD, Department of Medicine, Section of Hepatobiliary Diseases, University of Florida, 1600 South West Archer Road, PO Box 100214, Gainesville, Florida 32610 – 0214. © 1999 by Excerpta Medica, Inc. All rights reserved.
T
here are a variety of ways to categorize disease and the agents that cause them. Phenotype distinctions are based on the clinical presentation and behavior of a disease. Antibody neutralization of a specific antigen defines a serotype; this is typically used in distinguishing bacterial strains. Genotype and quasispecies refer to relatedness on a molecular or genetic level. Differences between genotypes are relatively large—there are nucleotide differences in 30% of the viral sequence. Quasispecies represent minor molecular variations, with only 1% or 2% nucleotide heterogeneity. The striking genetic heterogeneity of hepatitis C virus (HCV) is well recognized. This genetic diversity includes major genotypes (six), with numerous subtypes (over 80) and minor variants called “quasispecies.” The clinical implications of these variants, techniques for their identification, and their geographic distribution will be described.
MECHANISM OF GENOTYPE EVOLUTION During replication of the hepatitis C virus, RNA-dependent RNA polymerase frequently introduces random nucleotide errors. This results in a relatively high rate of spontaneous nucleotide substitutions, between 10⫺2 and 10⫺3/site/year. These replication errors yield 10 to 100 viable replacements per site per year. Whether a given nucleotide substitution results in a viable mutant is site dependent; different regions of the hepatitis C viral genome vary in their level of genetic conservation. Highly conserved areas include those coding for crucial functions, such as polymerase and other nonstructural proteins, core protein, the 5⬘ noncoding region, and the internal ribosome entry site. Nucleotide substitutions in these regions are less likely to be viable, because changes may result in altered structure that precludes function. Other regions of the genome are more tolerant of nucleotide substitutions, including the hypervariable regions. Such areas would be optimal for identifying subtle sequence variations, as in quasispecies analyses. The high rate of random nucleotide substitutions introduced by the viral RNA polymerase and the constant introduction of replication errors results in slow genetic evolution. As a consequence, HCV is a highly heterogeneous virus, with only approximately 70% homology between all isolates. The basis for classification into genotypes is genetic, or molecular, relatedness. Genotypes represent the molecular distance between different isolates of HCV. Because comparison of full0002-9343/99/$20.00 21S PII S002-9343(99)00376-9
A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis
Figure 1. Phylogenetic tree showing relatedness of six major hepatitis C genotypes and subtypes; produced by analysis of NS-5 sequences from 76 isolates of HCV. (Adapted with permission from Hepatology.1)
length viral sequences is impractical, several approaches have been developed to determine genetic relatedness of HCV isolates. Initially, investigators used direct sequencing of only the hypervariable region. Later, at least two regions were sequenced. Another method is phylogenetic tree analysis, which uses a variety of mathematical and statistical models to define genotype differences based on predicted changes within nucleotides. The more changes predicted, the greater the molecular distance between isolates. In 1993, Simmonds et al1 from Scotland published the phylogenetic tree analysis of the genotypes of HCV shown in Figure 1. It schematically shows the genetic distance between isolates. There are six major genotypes of HCV and at least 80 subtypes. Other genotypes have been defined, but subsequent evaluation has revealed that genotypes 7, 8, and 9 are actually subtypes of genotype 6.2 Further study of proposed genotype 10 showed that that is a subtype of genotype 3B. At this point, it is believed that there are six major genotypes of the HCV.
methods that we will be using clinically and are most familiar to us are the PCR-based approaches, which include sequencing, type specific primers, selective hybridization, which is the basis of the LiPA assay, and restriction fragment length polymorphism (RFLP). The LiPA test is based on differences coded by the NS4 region, and it detects genotypes 1 through 6. Overall, concordance between most of the different genotyping methods exceeds 93%.3 One exception is core-region genotype specific primers, the initial method described in Japan by Okamoto and colleagues.3a That technique is not as sensitive as others for the identification of genotype 1B. Both the RFLP4 and the LiPA technique that target the most highly conserved area of the HCV genome, the 5⬘ noncoding region, are sensitive, convenient, and economical. In addition, RFLP and LiPA results show high concordance with the more laborious sequencing and primerspecific PCR methods.3
HCV GENOTYPING ASSAYS
GEOGRAPHIC DISTRIBUTION OF HCV GENOTYPES
There are a variety of techniques for determining the genotype of an HCV isolate, which basically fall into two categories: PCR-based approaches and genotype-specific antibody approaches. These techniques identify different conserved portions of the HCV genome (Figure 1). The
Throughout the world, HCV genotypes differ with geographic region, as shown in Figure 2. HCV genotype 1 accounts for two thirds of HCV-infected individuals in the United States, with approximately equal numbers infected with genotypes 1A and 1B. Another 10% are in-
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Figure 2. Worldwide distribution of predominant hepatitis C genotypes.
fected with genotype 2, and 6% are infected with genotype 3. Approximately 10% are infected with mixed genotypes, and the genotype cannot be determined in the remainder.5 As in the United States, genotype 1 (specificially 1B) is predominant in Europe. Genotypes 2 and 3 are typically found in 15% to 30% of infected Europeans. Genotype 3 is very common in some parts of Europe; in Scandinavia, for example, 50% of HCV infections are genotype 3. Genotype 3 is also prevalent in the Far East (especially in Thailand), Pakistan, parts of India, and Australia. Genotype 4 is found almost exclusively in the Middle East. Genotype 5 is nearly always found in South Africa, and genotype 6 largely in Hong Kong.6,7
alcohol use, and response to therapy, were ignored.5,8 –11 Similarly, the impact of genotype on clinical and histologic behavior after liver transplantation has been extensively studied, with no definitive answers. Overall, there has been a suggestion in the literature that infection with genotype 1B is associated with more aggressive histology and greater recurrence after transplantation. Terrault et al12 analyzed HCV genotypes in clinic patients with liver disease and hepatic failure, and those that received liver transplants. Table 1 shows that similar proportions of patients with genotype 1 are seen in clinic and receive transplants, but proportionately more patients with genotype 2 and 3 receive liver transplants than are seen in clinic. These results suggest that genotype 1 is not associ-
IMPACT OF HCV GENOTYPE ON NATURAL HISTORY AND TREATMENT
Table 1. Impact of Hepatitis C Virus Genotype on Natural History
Whether HCV genotype affects the natural history and clinical behavior of hepatitis C has been debated in the literature from the time that genotypic distinctions were first noted. Many studies have focused on whether infection with genotype 1B leads to worse histology, but no consensus has been reached. In many studies, genotype was the only variable considered, and important other variables, including duration of infection, concomitant
Patients (%)* Genotype 1 2/3
Seen in Clinics
Received Transplant
67 16
61 25
* Patients at the University of California–San Francisco Transplant Center. Adapted from J Med Virol.12
December 27, 1999
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Figure 3. Response to therapy with ribavirin and interferon alfa-2b (Rebetron) or interferon alfa-2b alone (Intron A) in previously untreated patients according to hepatitis C genotype; m ⫽ months. (Adapted with permission from N Engl J Med.22)
ated with a worse prognosis.13–21 It is clear that more rigorous study is necessary. Genotype differences may be of considerable clinical importance if they are shown to reflect differences in response to therapy. McHutchison et al22 examined the impact of HCV genotype on response to combination therapy with ribavirin and interferon alfa-2b and interferon alone. Figure 3 shows the marked difference in response to therapy in treatment-naı¨ve patients infected with genotype 1 as compared with genotypes 2 and 3. Nearly 70% of patients with genotype 2 or 3 experienced sustained responses to combination therapy, even with the shorter duration of treatment (6 months). In comparison, 12 months of combination therapy led to a sustained response in only 30% of patients infected with genotype 1. Similar results were noted in patients who failed prior interferon therapy. Additionally, 6 months of combination therapy with ribavirin and interferon alfa-2b led to sustained responses in 75% of genotype 2 or 3 relapsers, but only in 30% of relapsers infected with HCV genotype 1.22 These results suggest that genotype influences response to therapy and indicate a need for development of aggressive therapies specifically directed toward resistant HCV genotype 1. Because no specific therapies for HCV genotype 1 exist and some people with genotype 1 do respond to therapy, genotyping to determine eligibility for treatment is not recommended.
QUASISPECIES The term “quasispecies” refers to pools of related genetic variants with only minor sequence differences (i.e., less than 5% of the viral genome). Although the differential 24S December 27, 1999 THE AMERICAN JOURNAL OF MEDICINE威
response to therapy according to HCV genotype indicates that genotype differences are clinically significant, the evidence to date is far less compelling for clinical implications of quasispecies differences. Thus, testing for quasispecies in an individual is not yet feasible in clinical practice. There are a few points regarding quasispecies that are worth mentioning, however. In a given inoculum, there may be a large number of quasispecies, but not all quasispecies are infectious. There is a range of replicative efficiency among quasispecies.23 The diversity of quasispecies in any individual is related to the size of the original inoculum.24 However, the evolution of quasispecies from a common inoculum varies with the host. Studies of HCV transmission from mother to child have demonstrated that the quasispecies that becomes most numerous in the child may not have been the most common one in the mother at the time of viral transmission. Host-mediated reasons for these differences may be related to humoral or cellular immune responses to the distinct viral variants. Quasispecies may differ in liver and plasma, as well.25 Future studies may demonstrate the clinical significance of this difference. Limited studies have indicated that quasispecies differ with respect to the ALT elevations they induce, and some data suggest that greater diversity in HCV quasispecies is correlated with more severe liver disease.26 –28 This is thought to be a result of an increased number of targets for cytotoxic T-lymphocyte attack in the liver, but this mechanism has not been confirmed.26,27 Quasispecies also appear to differ in their response to interferon-alfa therapy.2,24 Interferon therapy reduces heterogeneity as viral replication decreases.28 However, Volume 107 (6B)
A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis
characterization studies have shown that after interferon therapy, different HCV quasispecies may become dominant. Finally, it appears that patients with more than two major quasispecies may respond poorly to interferon therapy.28,29 The quasispecies profile also often changes after liver transplantation. This implies a role for the liver, as well as host immune responses, in selection of predominant quasispecies.30,31
SUMMARY Hepatitis C virus is a heterogeneous virus with a high mutation rate. Major variants with up to 30% sequence difference represent the six major genotypes; multiple subtypes exist within each genotype. Minor sequence variations, on the order of 1% to 2%, are common; these quasispecies exist as a highly related but heterogeneous pool of isolates. Genotypes clearly show differences in their geographic distribution, with type 1B most common in the United States. The impact of genotype on the natural history of hepatitis C virus infection is controversial, but recent data show that genotype impacts significantly on response to therapy. This may ultimately influence choice and duration of therapy and perhaps affect patient selection for treatment, but such recommendations do not yet exist.
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A Symposium: Hepatitis C Virus Genotypes and Quasispecies/Davis tion and responses to interferon alfa therapy in patients with chronic hepatitis C. J Med Virol. 1998;54:256 –264. 25. Maggi F, Fornai C, Vatteroni ML, et al. Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma. J Gen Virol. 1997;78:1521–1525. 26. Yuki N, Hayashi N, Moribe T, et al. Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies. Hepatology. 1997;25: 439 – 444. 27. Hayashi J, Kishihara Y, Yamaji K, et al. Hepatitis C viral quasispecies and liver damage in patients with chronic hepatitis C virus infection. Hepatology. 1997;25:697–701.
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28. Gonzalez-Peralta RP, Qian K, She YS, et al. Clinical implications of viral quasispecies in chronic hepatitis C. J Med Virol. 1996;49:242–247. 29. Toyoda H, Kumada T, Nakano S, et al. Quasispecies nature of hepatitis C virus and response to alpha interferon: significance as a predictor of direct response to interferon. J Hepatol. 1997;26:6 –13. 30. Yun Z, Barkholt L, Sonnerborg A. Dynamic analysis of hepatitis C virus polymorphism in patients with orthotopic liver transplantation. Transplantation. 1997;64:170 –172. 31. Lawal Z, Petrik J, Wong VS, et al. Hepatitis C virus genomic variation in untreated and immunosuppressed patients. Virology. 1997;228:107–111.
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