title: Comparison of amplification enzymes for Hepatitis C Virus quasispecies analysis.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA. polyak@u.washington.edu
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Polyak
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Sullivan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Austin
authors.forename: James Y
authors.initials: JY
authors.lastname: Dai
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Shuhart
authors.forename: Karen L
authors.initials: KL
authors.lastname: Lindsay
authors.forename: Herbert L
authors.initials: HL
authors.lastname: Bonkovsky
authors.forename: Adrian M
authors.initials: AM
authors.lastname: Di Bisceglie
authors.forename: William M
authors.initials: WM
authors.lastname: Lee
authors.forename: Chihiro
authors.initials: C
authors.lastname: Morishima
authors.forename: David R
authors.initials: DR
authors.lastname: Gretch
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000627:therapeutic use; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005260:Female; D014644:Genetic Variation; D015658:HIV Infections / Q000150:complications / Q000188:drug therapy; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase