A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells.
Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 microg liver RNA and 10(7)-10(8) copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10(9) copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.
Lin
Lan
L
Department of Medicine, Division of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium.
Fevery
Johan
J
Hiem Yap
Sing
S
D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D019698:Hepatitis C, Chronic / Q000097:blood / Q000821:virology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008099:Liver / Q000821:virology; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014779:Virus Replication
D012367:RNA, Viral