Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction.
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.
Hu
Yuan
Y
Microbiological Sciences Branch, Northeast Regional Laboratory, US Food and Drug Administration, Jamaica, NY, USA.
Shahidi
Azra
A
Park
Soon
S
Guilfoyle
Dennis
D
Hirshfield
Irvin
I
D001402:B-Lymphocytes / Q000821:virology; D017931:DNA Primers / Q000737:chemistry; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D007945:Leukemia, Lymphoid / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000821:virology; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000502:physiology*
D017931:DNA Primers; D012367:RNA, Viral