Peripheral blood mononuclear cells are possible extrahepatic replication sites for hepatitis C virus.
OBJECTIVE
Hepatitis C virus is a major causative agent of chronic liver disease and hepatocellular carcinoma and is considered to be a hepatotropic virus. It remains controversial whether hepatitis C virus exists in peripheral blood mononuclear cells and replicates there. In order to resolve this issue, we performed nested RT-PCR (reverse transcription polymerase chain reaction) and RT-PCR in situ hybridization in peripheral blood mononuclear cells of patients with chronic hepatitis C.


METHODS
We collected peripheral blood mononuclear cells from patients with chronic hepatitis C, extracted total RNA from the samples, and performed nested RT-PCR to detect hepatitis C virus RNA in the peripheral blood mononuclear cells lysates. We also fixed peripheral blood mononuclear cells of the patients in 4% paraformaldehyde and performed RT-PCR in situ hybridization with a digoxigenin-labeled RNA probe to detect hepatitis C virus RNA in the cells.


RESULTS
Using these methods, we detected both positive- and negative-stranded hepatitis C virus RNA in peripheral blood mononuclear cells of hepatitis C patients. To determine in which cell population of peripheral blood mononuclear cells hepatitis C virus is present, we performed PCR in situ hybridization after incubation with fluorescent latex microbeads which could be phagocytozed by monocytes. We obtained positive signals of the replicative hepatitis C virus genome not only in lymphocytes but also in monocytes.


CONCLUSIONS
RT-PCR in situ hybridization with a nonradioactive probe was found to be useful for in situ detection of hepatitis C virus RNA. Our findings suggest that peripheral blood mononuclear cells may be extrahepatic replication sites for hepatitis C virus.
Third Department of Internal Medicine, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
Shuhei
S
Nishiguchi
Katsuhiko
K
Fukuda
Susumu
S
Shiomi
Tadashi
T
Takeda
Tetsuo
T
Kuroki
Masayuki
M
Ogami
Hideki
H
Morimoto
Shuzo
S
Otani
Masami
M
Sakurai
Akio
A
Matsuhisa
D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication
D012367:RNA, Viral