Full-Length Open Reading Frame Amplification of Hepatitis C Virus

  • Ulrik Fahnøe
  • Jens Bukh
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


The purpose of this method is to amplify the full coding sequence of hepatitis C virus (HCV) by a single round reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Our method relies on a highly robust and sensitive RNA extraction procedure and cutting-edge RT-PCR enzymes, all of which have been rigorously tested and optimized. This will not only allow for robust amplification of the entire open reading frame (ORF) of HCV for sequencing by Sanger or next-generation sequencing (NGS), but can also be used for cloning of the ORF of uncharacterized samples and for linkage analysis of mutations on individual genomes spanning the entire ORF. The method has been validated on a variety of samples, including sera from HCV patients and cell-culture supernatants.

Key words

Hepatitis C virus HCV RNA extraction Long PCR Full-length ORF RT-PCR Next-generation sequencing Sanger sequencing Cloning 


  1. 1.
    Bukh J (2016) The history of hepatitis C virus (HCV): basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control. J Hepatol 65:S2–S21CrossRefGoogle Scholar
  2. 2.
    Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci 91:2216–2220CrossRefGoogle Scholar
  3. 3.
    Tellier R, Bukh J, Emerson SU et al (1996) Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes. J Clin Microbiol 34:3085–3091PubMedPubMedCentralGoogle Scholar
  4. 4.
    Yanagi M, Purcell RH, Emerson SU, Bukh J (1997) Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94:8738–8743CrossRefGoogle Scholar
  5. 5.
    Rasmussen TB, Reimann I, Hoffmann B et al (2008) Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon. J Virol Methods 149:330–333CrossRefGoogle Scholar
  6. 6.
    Fahnøe U, Pedersen AG, Dräger C et al (2015) Creation of functional viruses from non-functional cDNA clones obtained from an RNA virus population by the use of ancestral reconstruction. PLoS One 10:1–17CrossRefGoogle Scholar
  7. 7.
    Fahnøe U, Pedersen AG, Risager PC et al (2014) Rescue of the highly virulent classical swine fever virus strain “Koslov” from cloned cDNA and first insights into genome variations relevant for virulence. Virology 468:379–387CrossRefGoogle Scholar
  8. 8.
    Kostela J, Ayers M, Nishikawa J et al (2008) Amplification by long RT-PCR of near full-length norovirus genomes. J Virol Methods 149:226–230CrossRefGoogle Scholar
  9. 9.
    Li Y-P, Ramirez S, Jensen SB et al (2012) Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc Natl Acad Sci U S A 109:19757–19762CrossRefGoogle Scholar
  10. 10.
    Billerbeck E, Wolfisberg R, Fahnøe U et al (2017) Mouse models of acute and chronic hepacivirus infection. Science 357(80):204–208CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious DiseasesCopenhagen University HospitalHvidovreDenmark
  2. 2.Department of Immunology and Microbiology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

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