Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Jul;33(7):1870–1874. doi: 10.1128/jcm.33.7.1870-1874.1995

Dynamic analysis of heterogeneous hepatitis C virus populations by direct solid-phase sequencing.

J Odeberg 1, Z Yun 1, A Sönnerborg 1, M Uhlén 1, J Lundeberg 1
PMCID: PMC228288  PMID: 7665662

Abstract

In the present study, we used a semiautomated solid-phase direct sequencing method to analyze sequence diversity and variation of the hypervariable E2/NS1 region in the hepatitis C virus (HCV) genome in isolates from patients seropositive for HCV. A total of 24 isolates of various origins were sequenced. Six of the patients, not subject to any antiviral therapy, were monitored longitudinally, and rapid sequence variations were observed over a period of 14 months. The nucleotide change rate was found to be 0.1 to 0.2 nucleotide substitution per genome site per year. Furthermore, isolates from five of the patients were used for a comparative study of the direct solid-phase sequencing approach versus the frequently used approach of sequencing individual reverse transcriptase PCR clones. The advantage of direct solid-phase sequencing for studying dynamic changes in heterogeneous populations of HCV is discussed.

Full Text

The Full Text of this article is available as a PDF (197.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  2. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  3. Enomoto N., Sato C., Kurosaki M., Marumo F. Hepatitis C virus after interferon treatment has the variation in the hypervariable region of envelope 2 gene. J Hepatol. 1994 Feb;20(2):252–261. doi: 10.1016/s0168-8278(05)80066-2. [DOI] [PubMed] [Google Scholar]
  4. Higashi Y., Kakumu S., Yoshioka K., Wakita T., Mizokami M., Ohba K., Ito Y., Ishikawa T., Takayanagi M., Nagai Y. Dynamics of genome change in the E2/NS1 region of hepatitis C virus in vivo. Virology. 1993 Dec;197(2):659–668. doi: 10.1006/viro.1993.1641. [DOI] [PubMed] [Google Scholar]
  5. Hultman T., Bergh S., Moks T., Uhlén M. Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. Biotechniques. 1991 Jan;10(1):84–93. [PubMed] [Google Scholar]
  6. Kato N., Ootsuyama Y., Tanaka T., Nakagawa M., Nakazawa T., Muraiso K., Ohkoshi S., Hijikata M., Shimotohno K. Marked sequence diversity in the putative envelope proteins of hepatitis C viruses. Virus Res. 1992 Feb;22(2):107–123. doi: 10.1016/0168-1702(92)90038-b. [DOI] [PubMed] [Google Scholar]
  7. Kurosaki M., Enomoto N., Marumo F., Sato C. Rapid sequence variation of the hypervariable region of hepatitis C virus during the course of chronic infection. Hepatology. 1993 Dec;18(6):1293–1299. [PubMed] [Google Scholar]
  8. Leitner T., Halapi E., Scarlatti G., Rossi P., Albert J., Fenyö E. M., Uhlén M. Analysis of heterogeneous viral populations by direct DNA sequencing. Biotechniques. 1993 Jul;15(1):120–127. [PubMed] [Google Scholar]
  9. Okamoto H., Tokita H., Sakamoto M., Horikita M., Kojima M., Iizuka H., Mishiro S. Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection. J Gen Virol. 1993 Nov;74(Pt 11):2385–2390. doi: 10.1099/0022-1317-74-11-2385. [DOI] [PubMed] [Google Scholar]
  10. Sakamoto N., Enomoto N., Kurosaki M., Marumo F., Sato C. Sequential change of the hypervariable region of the hepatitis C virus genome in acute infection. J Med Virol. 1994 Jan;42(1):103–108. doi: 10.1002/jmv.1890420119. [DOI] [PubMed] [Google Scholar]
  11. Simmonds P., Holmes E. C., Cha T. A., Chan S. W., McOmish F., Irvine B., Beall E., Yap P. L., Kolberg J., Urdea M. S. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol. 1993 Nov;74(Pt 11):2391–2399. doi: 10.1099/0022-1317-74-11-2391. [DOI] [PubMed] [Google Scholar]
  12. Wahlberg J., Albert J., Lundeberg J., Cox S., Wahren B., Uhlén M. Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients. FASEB J. 1992 Jul;6(10):2843–2847. doi: 10.1096/fasebj.6.10.1634047. [DOI] [PubMed] [Google Scholar]
  13. Wahlberg J., Albert J., Lundeberg J., Von Gegerfelt A., Broliden K., Utter G., Fenyö E. M., Uhlén M. Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing. AIDS Res Hum Retroviruses. 1991 Dec;7(12):983–990. doi: 10.1089/aid.1991.7.983. [DOI] [PubMed] [Google Scholar]
  14. Yun Z. B., Lindh G., Weiland O., Johansson B., Sönnerborg A. Detection of hepatitis C virus (HCV) RNA by PCR related to HCV antibodies in serum and liver histology in Swedish blood donors. J Med Virol. 1993 Jan;39(1):57–61. doi: 10.1002/jmv.1890390111. [DOI] [PubMed] [Google Scholar]
  15. van Doorn L. J., Quint W., Tsiquaye K., Voermans J., Paelinck D., Kos T., Maertens G., Schellekens H., Murray K. Longitudinal analysis of hepatitis C virus infection and genetic drift of the hypervariable region. J Infect Dis. 1994 Jun;169(6):1226–1235. doi: 10.1093/infdis/169.6.1226. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES