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Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis

Show simple item record Polyak, Stephen J. en_US Sullivan, Daniel G. en_US Austin, Michael A. en_US Dai, James Y. en_US Shuhart, Margaret C. en_US Lindsay, Karen L. en_US Bonkovsky, Herbert L. en_US Di Bisceglie, Adrian M. en_US Lee, William M. en_US Morishima, Chihiro en_US Gretch, David R. en_US HALT-C Trial Group en_US 2010-05-06T20:03:16Z 2010-05-06T20:03:16Z 2005 en_US
dc.identifier.citation Polyak S, Sullivan D, Austin M, et al. Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis. Virology Journal. 2005;2(1):41. en_US
dc.identifier.other 10.1186/1743-422X-2-41 en_US
dc.identifier.uri en_US
dc.description.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 less than 0.0001, and r = 0.47; p less than 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. en_US
dc.description.sponsorship National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Allergy and Infectious Diseases, National Cancer Institute, National Center for Minority Health and Health Disparities, General Clinical Research Center. en_US
dc.language.iso en_US en_US
dc.title Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis en_US
dc.type Article en_US

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