Abstract
Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 105 IU/ml, 15 of 16 (94%) at 104 to 105 IU/ml, 6 of 7 (86%) at 103 to 104 IU/ml, and 2 of 3 (67%) at less than 103 IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated daring amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4223-4229 |
| Number of pages | 7 |
| Journal | Journal of Clinical Microbiology |
| Volume | 42 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 2004 |
Fingerprint
ASJC Scopus subject areas
- Microbiology (medical)
- Microbiology
Cite this
Accurate representation of the hepatitis C virus quasispecies in 5.2-kilobase amplicons. / Liu, Zhi; Netski, Dale M.; Mao, Qing; Laeyendecker, Oliver B.; Ticehurst, John R; Wang, Xiao Hong; Thomas, David L; Ray, Stuart Campbell.
In: Journal of Clinical Microbiology, Vol. 42, No. 9, 09.2004, p. 4223-4229.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Accurate representation of the hepatitis C virus quasispecies in 5.2-kilobase amplicons
AU - Liu, Zhi
AU - Netski, Dale M.
AU - Mao, Qing
AU - Laeyendecker, Oliver B.
AU - Ticehurst, John R
AU - Wang, Xiao Hong
AU - Thomas, David L
AU - Ray, Stuart Campbell
PY - 2004/9
Y1 - 2004/9
N2 - Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 105 IU/ml, 15 of 16 (94%) at 104 to 105 IU/ml, 6 of 7 (86%) at 103 to 104 IU/ml, and 2 of 3 (67%) at less than 103 IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated daring amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.
AB - Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 105 IU/ml, 15 of 16 (94%) at 104 to 105 IU/ml, 6 of 7 (86%) at 103 to 104 IU/ml, and 2 of 3 (67%) at less than 103 IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated daring amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.
UR - http://www.scopus.com/inward/record.url?scp=4644279066&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4644279066&partnerID=8YFLogxK
U2 - 10.1128/JCM.42.9.4223-4229.2004
DO - 10.1128/JCM.42.9.4223-4229.2004
M3 - Article
C2 - 15365015
AN - SCOPUS:4644279066
VL - 42
SP - 4223
EP - 4229
JO - Journal of Clinical Microbiology
JF - Journal of Clinical Microbiology
SN - 0095-1137
IS - 9
ER -