In vitro phenotyping of recombinant hepatitis B virus containing the polymerase/reverse transcriptase gene from clinical isolates

Methods Mol Biol. 2013:1030:163-81. doi: 10.1007/978-1-62703-484-5_14.

Abstract

This chapter describes the procedures for the generation of recombinant hepatitis B virus (HBV) that contains the polymerase/reverse transcriptase (pol/RT) gene from clinical isolates. It also contains procedures for phenotypic testing of the recombinant HBV in HepG2 cells for susceptibility to nucleos(t)ide analogs. Amplification and cloning of the pol/RT gene as opposed to the full-length HBV genome from a clinical isolate allows for clinical samples with lower viral loads to be successfully analyzed. Furthermore, it enables a direct comparison of sequence changes in the pol/RT region between isolates.

MeSH terms

  • Cell Culture Techniques
  • Cloning, Molecular
  • DNA Replication
  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification
  • Genes, pol*
  • Genetic Vectors / genetics
  • Genetic Vectors / isolation & purification
  • Hep G2 Cells
  • Hepatitis B virus / isolation & purification
  • Hepatitis B virus / physiology*
  • Humans
  • Multiplex Polymerase Chain Reaction
  • Phenotype*
  • Plasmids / genetics
  • Plasmids / isolation & purification
  • Polymerase Chain Reaction
  • RNA-Directed DNA Polymerase / genetics*
  • Real-Time Polymerase Chain Reaction
  • Recombination, Genetic*
  • Virus Replication

Substances

  • DNA, Viral
  • RNA-Directed DNA Polymerase