This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, Y.-M.
Right arrow Articles by Thomas, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, Y.-M.
Right arrow Articles by Thomas, D. L.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 1998, p. 2982-2989, Vol. 36, No. 10
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Assessment of Hepatitis C Virus Sequence Complexity by Electrophoretic Mobilities of Both Single-and Double-Stranded DNAs

Yu-Ming Wang,1 Stuart C. Ray,1 Oliver Laeyendecker,1 John R. Ticehurst,2,3 and David L. Thomas1,*

Departments of Medicine1 and Pathology,2 Johns Hopkins University School of Medicine, Baltimore, and Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville,3 Maryland

Received 4 May 1998/Returned for modification 15 June 1998/Accepted 16 July 1998

To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean ± standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% ± 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% ± 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% ± 1.8% and 3.5% ± 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (R2, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.

* Corresponding author. Mailing address: Division of Infectious Diseases, 720 Rutland Ave., Ross 1159, Baltimore, MD 21205. Phone: (410) 955-0349. Fax: (410) 955-7889. E-mail: dthomas{at}welchlink.welch.jhu.edu.

Journal of Clinical Microbiology, October 1998, p. 2982-2989, Vol. 36, No. 10
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Ferreira, C., Singh, Y., Furmanski, A. L., Wong, F. S., Garden, O. A., Dyson, J. (2009). Non-obese diabetic mice select a low-diversity repertoire of natural regulatory T cells. Proc. Natl. Acad. Sci. USA 106: 8320-8325 [Abstract] [Full Text]  
  • Wolfl, M., Rutebemberwa, A., Mosbruger, T., Mao, Q., Li, H.-m., Netski, D., Ray, S. C., Pardoll, D., Sidney, J., Sette, A., Allen, T., Kuntzen, T., Kavanagh, D. G., Kuball, J., Greenberg, P. D., Cox, A. L. (2008). Hepatitis C Virus Immune Escape via Exploitation of a Hole in the T Cell Repertoire. J. Immunol. 181: 6435-6446 [Abstract] [Full Text]  
  • Wang, X.-H., Netski, D. M., Astemborski, J., Mehta, S. H., Torbenson, M. S., Thomas, D. L., Ray, S. C. (2007). Progression of Fibrosis during Chronic Hepatitis C Is Associated with Rapid Virus Evolution. J. Virol. 81: 6513-6522 [Abstract] [Full Text]  
  • Cox, A. L., Mosbruger, T., Mao, Q., Liu, Z., Wang, X.-H., Yang, H.-C., Sidney, J., Sette, A., Pardoll, D., Thomas, D. L., Ray, S. C. (2005). Cellular immune selection with hepatitis C virus persistence in humans. JEM 201: 1741-1752 [Abstract] [Full Text]  
  • Herring, B. L., Tsui, R., Peddada, L., Busch, M., Delwart, E. L. (2005). Wide Range of Quasispecies Diversity during Primary Hepatitis C Virus Infection. J. Virol. 79: 4340-4346 [Abstract] [Full Text]  
  • Mas, A., Ulloa, E., Bruguera, M., Furcic, I., Garriga, D., Fabregas, S., Andreu, D., Saiz, J. C., Diez, J. (2004). Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity. J. Gen. Virol. 85: 3619-3626 [Abstract] [Full Text]  
  • Liu, Z., Netski, D. M., Mao, Q., Laeyendecker, O., Ticehurst, J. R., Wang, X.-H., Thomas, D. L., Ray, S. C. (2004). Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons. J. Clin. Microbiol. 42: 4223-4229 [Abstract] [Full Text]  
  • Tsui, R., Herring, B. L., Barbour, J. D., Grant, R. M., Bacchetti, P., Kral, A., Edlin, B. R., Delwart, E. L. (2004). Human Immunodeficiency Virus Type 1 Superinfection Was Not Detected following 215 Years of Injection Drug User Exposure. J. Virol. 78: 94-103 [Abstract] [Full Text]  
  • Thimme, R., Oldach, D., Chang, K.-M., Steiger, C., Ray, S. C., Chisari, F. V. (2001). Determinants of Viral Clearance and Persistence during Acute Hepatitis C Virus Infection. JEM 194: 1395-1406 [Abstract] [Full Text]  
  • Mao, Q., Ray, S. C., Laeyendecker, O., Ticehurst, J. R., Strathdee, S. A., Vlahov, D., Thomas, D. L. (2001). Human Immunodeficiency Virus Seroconversion and Evolution of the Hepatitis C Virus Quasispecies. J. Virol. 75: 3259-3267 [Abstract] [Full Text]  
  • Oldach, D. W., Delwiche, C. F., Jakobsen, K. S., Tengs, T., Brown, E. G., Kempton, J. W., Schaefer, E. F., Bowers, H. A., Glasgow, H. B. Jr., Burkholder, J. M., Steidinger, K. A., Rublee, P. A. (2000). Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools. Proc. Natl. Acad. Sci. USA 97: 4303-4308 [Abstract] [Full Text]  
  • Ray, S. C., Mao, Q., Lanford, R. E., Bassett, S., Laeyendecker, O., Wang, Y.-M., Thomas, D. L. (2000). Hypervariable Region 1 Sequence Stability during Hepatitis C Virus Replication in Chimpanzees. J. Virol. 74: 3058-3066 [Abstract] [Full Text]  
  • Sandres, K., Dubois, M., Pasquier, C., Payen, J. L., Alric, L., Duffaut, M., Vinel, J. P., Pascal, J. P., Puel, J., Izopet, J. (2000). Genetic Heterogeneity of Hypervariable Region 1 of the Hepatitis C Virus (HCV) Genome and Sensitivity of HCV to Alpha Interferon Therapy. J. Virol. 74: 661-668 [Abstract] [Full Text]  
  • Ray, S. C., Wang, Y.-M., Laeyendecker, O., Ticehurst, J. R., Villano, S. A., Thomas, D. L. (1999). Acute Hepatitis C Virus Structural Gene Sequences as Predictors of Persistent Viremia: Hypervariable Region 1 as a Decoy. J. Virol. 73: 2938-2946 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»