OUP user menu

Detection of Extrahepatic Hepatitis C Virus Replication by a Novel, Highly Sensitive, Single-Tube Nested Polymerase Chain Reaction

Yuan Hu MD, Azra Shahidi PhD, Soon Park, Dennis Guilfoyle PhD, Irvin Hirshfield PhD
DOI: http://dx.doi.org/10.1309/33TAJLB748KLMXVG 95-100 First published online: 1 January 2003


We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Key Words:
  • Hepatitis C virus
  • Lymphocyte
  • Complementary DNA
  • PCR
  • Polymerase chain reaction
  • Nested PCR
  • DNA sequencing