Open Access Research

Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis

Stephen J Polyak123*, Daniel G Sullivan1, Michael A Austin1, James Y Dai4, Margaret C Shuhart5, Karen L Lindsay7, Herbert L Bonkovsky8, Adrian M Di Bisceglie9, William M Lee10, Chihiro Morishima16, David R Gretch15 and the HALT-C Trial Group

Author Affiliations

1 Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA

2 Department of Microbiology, University of Washington, Seattle, WA, USA

3 Department of Pathobiology, University of Washington, Seattle, WA, USA

4 Department of Biostatistics, University of Washington, Seattle, WA, USA

5 Department of Medicine, University of Washington, Seattle, WA, USA

6 Department of Pediatrics, University of Washington, Seattle, WA, USA

7 Division of Gastrointestinal and Liver Diseases, University of Southern California, Los Angeles, CA, USA

8 Liver-Biliary-Pancreatic Center and the General Clinical Research Center, University of Connecticut Health Center, Farmington, CT, USA

9 Division of Gastroenterology and Hepatology, Saint Louis University School of Medicine, St. Louis, MO, USA

10 Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, TX, USA

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Virology Journal 2005, 2:41  doi:10.1186/1743-422X-2-41

Published: 22 April 2005

Abstract

Background

Hepatitis C virus (HCV) circulates as quasispecies (QS), whose evolution is associated with pathogenesis. Previous studies have suggested that the use of thermostable polymerases without proofreading function may contribute to inaccurate assessment of HCV QS. In this report, we compared non-proofreading (Taq) with proofreading (Advantage High Fidelity-2; HF-2) polymerases in the sensitivity, robustness, and HCV QS diversity and complexity in the second envelope glycoprotein gene hypervariable region 1 (E2-HVR1) on baseline specimens from 20 patients in the HALT-C trial and in a small cohort of 12 HCV/HIV co-infected patients. QS diversity and complexity were quantified using heteroduplex mobility assays (HMA).

Results

The sensitivities of both enzymes were comparable at 50 IU/ml, although HF-2 was more robust and slightly more sensitive than Taq. Both enzymes generated QS diversity and complexity scores that were correlated (r = 0.68; p < 0.0001, and r = 0.47; p < 0.01; Spearman's rank correlation). QS diversity was similar for both Taq and HF-2 enzymes, although there was a trend for higher diversity in samples amplified by Taq (p = 0.126). Taq amplified samples yielded complexity scores that were significantly higher than HF-2 samples (p = 0.033). HALT-C patients who were HCV positive or negative following 20 weeks of pegylated IFN plus ribavirin therapy had similar QS diversity scores for Taq and HF-2 samples, and there was a trend for higher complexity scores from Taq as compared with HF-2 samples. Among patients with HCV and HIV co-infection, HAART increased HCV QS diversity and complexity as compared with patients not receiving therapy, suggesting that immune reconstitution drives HCV QS evolution. However, diversity and complexity scores were similar for both HF-2 and Taq amplified specimens.

Conclusion

The data suggest that while Taq may overestimate HCV QS complexity, its use does not significantly affect results in cohort-based studies of HCV QS analyzed by HMA. However, the use of proofreading enzymes such as HF-2 is recommended for more accurate characterization of HCV QS in vivo.

Keywords:
hepatitis C virus; HALT-C; quasispecies; hypervariable region; E2