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. 1991 Dec;29(12):2860–2864. doi: 10.1128/jcm.29.12.2860-2864.1991

Discrimination of hepatitis C virus in liver tissues from different patients with hepatocellular carcinomas by direct nucleotide sequencing of amplified cDNA of the viral genome.

W H Chou 1, T Yoneyama 1, K Takeuchi 1, H Harada 1, I Saito 1, T Miyamura 1
PMCID: PMC270447  PMID: 1661747

Abstract

We investigated the presence of the hepatitis C virus (HCV) genome in liver tissues of eight different patients with hepatocellular carcinoma by using the reverse transcriptase polymerase chain reaction (PCR) method. RNA was extracted separately from cancerous and peripheral noncancerous portions of the liver tissues of each patient. For reverse transcriptase PCR, we used sets of primers derived either from nonstructural region 3 (the NS3 region) or from the nucleocapsid-envelope (C/E) region of the HCV genome. The nucleotide sequences of the amplimers were directly determined without subcloning. Of 16 samples tested, cDNA of the HCV genome was detected in 2 cancerous tissues and in 4 noncancerous tissues by either pair of primers. Nucleotide sequences of HCV cDNA fragments amplified from cancerous and peripheral noncancerous tissues from the same patients were identical. However, 4.4 to 6.3% and 7.5 to 11.3% sequence variation was observed in NS3 and C/E regions, respectively, among cDNA fragments from different patients. The result indicated that the HCV genome detected in a given patient is distinguishable from that in others by a simple direct nucleotide sequencing of the reverse transcriptase PCR products.

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Selected References

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

  1. Alter H. J., Purcell R. H., Shih J. W., Melpolder J. C., Houghton M., Choo Q. L., Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med. 1989 Nov 30;321(22):1494–1500. doi: 10.1056/NEJM198911303212202. [DOI] [PubMed] [Google Scholar]
  2. Anstee D. J. Blood group-active surface molecules of the human red blood cell. Vox Sang. 1990;58(1):1–20. doi: 10.1111/j.1423-0410.1990.tb02049.x. [DOI] [PubMed] [Google Scholar]
  3. Bruix J., Barrera J. M., Calvet X., Ercilla G., Costa J., Sanchez-Tapias J. M., Ventura M., Vall M., Bruguera M., Bru C. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet. 1989 Oct 28;2(8670):1004–1006. doi: 10.1016/s0140-6736(89)91015-5. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colombo M., Kuo G., Choo Q. L., Donato M. F., Del Ninno E., Tommasini M. A., Dioguardi N., Houghton M. Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet. 1989 Oct 28;2(8670):1006–1008. doi: 10.1016/s0140-6736(89)91016-7. [DOI] [PubMed] [Google Scholar]
  7. Enomoto N., Takada A., Nakao T., Date T. There are two major types of hepatitis C virus in Japan. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1021–1025. doi: 10.1016/0006-291x(90)90494-8. [DOI] [PubMed] [Google Scholar]
  8. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Ohkoshi S., Shimotohno K. Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun. 1991 Feb 28;175(1):220–228. doi: 10.1016/s0006-291x(05)81223-9. [DOI] [PubMed] [Google Scholar]
  10. Kaneko S., Unoura M., Kobayashi K., Kuno K., Murakami S., Hattori N. Detection of serum hepatitis C virus RNA. Lancet. 1990 Apr 21;335(8695):976–976. doi: 10.1016/0140-6736(90)91042-9. [DOI] [PubMed] [Google Scholar]
  11. Katayama T., Kikuchi S., Tanaka Y., Saito I., Miyamura T., Choo Q. L., Houghton M., Kuo G. Blood screening for non-A, non-B hepatitis by hepatitis C virus antibody assay. Transfusion. 1990 May;30(4):374–376. doi: 10.1046/j.1537-2995.1990.30490273448.x. [DOI] [PubMed] [Google Scholar]
  12. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kew M. C., Houghton M., Choo Q. L., Kuo G. Hepatitis C virus antibodies in southern African blacks with hepatocellular carcinoma. Lancet. 1990 Apr 14;335(8694):873–874. doi: 10.1016/0140-6736(90)90474-j. [DOI] [PubMed] [Google Scholar]
  14. Kubo Y., Takeuchi K., Boonmar S., Katayama T., Choo Q. L., Kuo G., Weiner A. J., Bradley D. W., Houghton M., Saito I. A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucleic Acids Res. 1989 Dec 25;17(24):10367–10372. doi: 10.1093/nar/17.24.10367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuo G., Choo Q. L., Alter H. J., Gitnick G. L., Redeker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989 Apr 21;244(4902):362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  16. Maéno M., Kaminaka K., Sugimoto H., Esumi M., Hayashi N., Komatsu K., Abe K., Sekiguchi S., Yano M., Mizuno K. A cDNA clone closely associated with non-A, non-B hepatitis. Nucleic Acids Res. 1990 May 11;18(9):2685–2689. doi: 10.1093/nar/18.9.2685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miyamura T., Saito I., Katayama T., Kikuchi S., Tateda A., Houghton M., Choo Q. L., Kuo G. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: application to diagnosis and blood screening for posttransfusion hepatitis. Proc Natl Acad Sci U S A. 1990 Feb;87(3):983–987. doi: 10.1073/pnas.87.3.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nishioka K., Watanabe J., Furuta S., Tanaka E., Iino S., Suzuki H., Tsuji T., Yano M., Kuo G., Choo Q. L. A high prevalence of antibody to the hepatitis C virus in patients with hepatocellular carcinoma in Japan. Cancer. 1991 Jan 15;67(2):429–433. doi: 10.1002/1097-0142(19910115)67:2<429::aid-cncr2820670218>3.0.co;2-#. [DOI] [PubMed] [Google Scholar]
  19. Ogata N., Alter H. J., Miller R. H., Purcell R. H. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3392–3396. doi: 10.1073/pnas.88.8.3392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohkoshi S., Kojima H., Tawaraya H., Miyajima T., Kamimura T., Asakura H., Satoh A., Hirose S., Hijikata M., Kato N. Prevalence of antibody against non-A, non-B hepatitis virus in Japanese patients with hepatocellular carcinoma. Jpn J Cancer Res. 1990 Jun-Jul;81(6-7):550–553. doi: 10.1111/j.1349-7006.1990.tb02605.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Okamoto H., Okada S., Sugiyama Y., Yotsumoto S., Tanaka T., Yoshizawa H., Tsuda F., Miyakawa Y., Mayumi M. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990 Jun;60(3):167–177. [PubMed] [Google Scholar]
  22. Okayama H., Kawaichi M., Brownstein M., Lee F., Yokota T., Arai K. High-efficiency cloning of full-length cDNA; construction and screening of cDNA expression libraries for mammalian cells. Methods Enzymol. 1987;154:3–28. doi: 10.1016/0076-6879(87)54067-8. [DOI] [PubMed] [Google Scholar]
  23. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  24. Saito I., Miyamura T., Ohbayashi A., Harada H., Katayama T., Kikuchi S., Watanabe Y., Koi S., Onji M., Ohta Y. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6547–6549. doi: 10.1073/pnas.87.17.6547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sbolli G., Zanetti A. R., Tanzi E., Cavanna L., Civardi G., Fornari F., Di Stasi M., Buscarini L. Serum antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. J Med Virol. 1990 Mar;30(3):230–232. doi: 10.1002/jmv.1890300316. [DOI] [PubMed] [Google Scholar]
  27. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takeuchi K., Boonmar S., Kubo Y., Katayama T., Harada H., Ohbayashi A., Choo Q. L., Kuo G., Houghton M., Saito I. Hepatitis C viral cDNA clones isolated from a healthy carrier donor implicated in post-transfusion non-A, non-B hepatitis. Gene. 1990 Jul 16;91(2):287–291. doi: 10.1016/0378-1119(90)90102-w. [DOI] [PubMed] [Google Scholar]
  29. Takeuchi K., Kubo Y., Boonmar S., Watanabe Y., Katayama T., Choo Q. L., Kuo G., Houghton M., Saito I., Miyamura T. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol. 1990 Dec;71(Pt 12):3027–3033. doi: 10.1099/0022-1317-71-12-3027. [DOI] [PubMed] [Google Scholar]
  30. Weiner A. J., Brauer M. J., Rosenblatt J., Richman K. H., Tung J., Crawford K., Bonino F., Saracco G., Choo Q. L., Houghton M. Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins. Virology. 1991 Feb;180(2):842–848. doi: 10.1016/0042-6822(91)90104-j. [DOI] [PubMed] [Google Scholar]
  31. Weiner A. J., Kuo G., Bradley D. W., Bonino F., Saracco G., Lee C., Rosenblatt J., Choo Q. L., Houghton M. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990 Jan 6;335(8680):1–3. doi: 10.1016/0140-6736(90)90134-q. [DOI] [PubMed] [Google Scholar]
  32. Yoneyama T., Takeuchi K., Watanabe Y., Harada H., Ohbayashi A., Tanaka Y., Yuasa T., Saito I., Miyamura T. Detection of hepatitis C virus cDNA sequence by the polymerase chain reaction in hepatocellular carcinoma tissues. Jpn J Med Sci Biol. 1990 Jun;43(3):89–94. doi: 10.7883/yoken1952.43.89. [DOI] [PubMed] [Google Scholar]

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