Quasispecies of Hepatitis C Virus

* To define quasispecies and consider the potential biological consequences of quasispecies development
* To review the molecular methods used to detect and quantify hepatitis C virus (HCV) quasispecies
* To examine the role of HCV quasispecies in determining (i) severity of disease, and (ii) response to interferon

Abstract:

The RNA-dependent polymerase of RNA viruses is highly error prone and lacking in proofreading capabilities. Consequently, within an individual, HCV exists as a heterogeneous mixture of closely related viruses called quasispecies. In contrast to HCV genotypes which vary by 31% to 35% of bases over the entire length of the genome, quasispecies vary from each other by 1% to 9% of bases.1 The quasispecies nature of HCV has several potentially important biological consequences. They are likely an important factor in the inability of acutely infected individuals to clear infection. Additionally, mutations in the viral populations likely contribute to drug “resistance” during interferon treatment and to the ineffectiveness of isolate-specific vaccines.

There are both direct and indirect methods of detecting and quantifying quasispecies within an individual. Early studies utilized cloned PCR products, a procedure which is relatively easy to perform and reliable, but labor-intensive. Indirect methods of measuring the number of different viral populations within an individual include single-strand conformation polymorphism (SSCP) and heteroduplex gel shift analysis (HDA).2,3 Using sequencing data as the “gold standard”, SSCP and HDA methods have been found to be sensitive enough to detect minor populations of virus (i.e., populations making up 5% or more of the total viral population) and viral isolates with three or more nucleotide changes. For nucleotide changes of only one to two base pairs, sequencing methods are more accurate than indirect methods. In the majority of clinical studies, the hypervariable region of the envelope region has been used to detect and quantify quasispecies.

Viral populations within an individual change over time.4 Greater viral diversity has been associated with more advanced stages of liver disease, although no causal relationship has been established.5 In immunosuppressed liver transplant recipients, emergence of minor quasispecies after transplantation has been linked to less aggressive post-transplant disease.3 The natural evolution of quasispecies in different patient populations is likely dependent upon the intensity of the immunological response, viral replication rates, and mutational frequencies over time.

The most relevant clinical application of quasispecies measurement relates to predicting interferon (IFN) responsiveness in patients with HCV genotype 1b.6 Japanese investigators have identified a region within the NS5A region (amino acids 2209-2248), termed the “interferon sensitivity determining region” or ISDR, which appears to determine response to IFN. In responders, four or more mutations were found within the ISDR, whereas non-responders had no mutations in the ISDR. The correlation between number of mutations within the ISDR and IFN response, however, was not absolute and thus other factors may be important. Additional studies are needed to determine the relationships between mutations in the ISDR and viral titer, treatment factors such as dose and duration of interferon, and host factors such duration of disease.

Norah Terrault, MD
Research Associate, Department of Medicine/Gastroenterology
University of California, San Francisco
River Center, Veteran’s Administration Medical Center
San Francisco, California

References:

1. Bukh J, Miller R, Purcell R. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Sem Liver Dis 1995;15(1):41-63.

2. Koizumi K, Enomoto N, Kurosaki M, et al. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. Hepatology 1995;22(1):30-5.

3. Gretch D, Polyak S, Wilson J, Carithers R, Perkins J, Corey L. Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients. J Virol 1996;70(11):7622-31.

4. Kurosaki M, Enomoto N, Marumo F, Sato C. Evolution and selection of hepatitis C virus variants in patients with chronic hepatitis C. Virology 1994;205:161-9.

5. Honda M, Kaneko S, Sakai A, Unoura M, Murakami S, Kobayashi K. Degree of diversity of hepatitis C virus quasispecies and progression of liver disease. Hepatology 1994;20(5):1144-51.

6. Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Eng J Med 1996;334(2):77-81.

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