Author for correspondence: Peter Simmonds. Fax +44 131 650 3138. e-mail Peter.Simmonds!ed.ac.uk Presented at the 4th International Meeting on Hepatitis C Virus and Related Viruses, March 1997, Kyoto, Japan (Abstract 128).
To investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 × 106 RNA copies per 106 cells and 3·7–4·2 × 103 copies per 106 cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7·3 × 103 per 106 cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4 , CD8 and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 × 103 copies per 106 cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.
BouffardP.,
HayashiP. H.,
AcevedoR.,
LevyN.,
ZeldisJ. B.1992; Hepatitis C virus is detected in a monocyte macrophage subpopulation of peripheral blood mononuclear cells of infected patients. Journal of Infectious Diseases 166:1276–1280
ChanS.-W.,
McOmishF.,
HolmesE. C.,
DowB.,
PeuthererJ. F.,
FollettE.,
YapP. L.,
SimmondsP.1992; Analysis of a new hepatitis C virus type and its phylogenetic relationship to existing variants. Journal of General Virology 73:1131–1141
ChavesR. L.,
GraffJ.,
NormannA.,
FlehmingB.1994; Specific detection of minus strand hepatitis A virus RNA by tail-PCR following reverse transcription. Nucleic Acids Research 22:1919–1920
DavidsonF.,
SimmondsP.,
FergusonJ. C.,
JarvisL. M.,
DowB. C.,
FollettE. A. C.,
SeedC. R. G.,
KrusiusT.,
LinC.,
MedgyesiG. A.,
KiyokawaH.,
OlimG.,
DuraisamyG.,
CuypersT.,
SaeedA. A.,
TeoD.,
ConradieJ.,
KewM. C.,
LinM.,
NuchaprayoonC.,
NdimbieO. K.,
YapP. L.1995; Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5′ noncoding region. Journal of General Virology 76:1197–1204
FongT. L.,
ShindoM.,
FeinstoneS. M.,
HoofnagleJ. H.,
Di BisceglieA. M.1991; Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C. Journal of Clinical Investigation 88:1058–1060
GabrielliA.,
ManzinA.,
CandelaM.,
CanigliaM. L.,
PaolucciS.,
DanieliM. G.,
ClementiM.1994; Active hepatitis C virus infection in bone marrow and peripheral blood mononuclear cells from patients with mixed cryoglobulinaemia. Clinical and Experimental Immunology 97:87–93
GilB.,
QianC.,
RiezubojJ. I.,
CiveiraM. P.,
PrietoJ.1993; Hepatic and extrahepatic HCV RNA strands in chronic hepatitis C - different patterns of response to interferon treatment. Hepatology 18:1050–1054
GunjiT.,
KatoN.,
HijikataM.,
HayashiK.,
SaitohS.,
ShimotohnoK.1994; Specific detection of positive and negative stranded hepatitis C viral RNA using chemical RNA modification. Archives of Virology 134:293–302
HawkinsA.,
DavidsonF.,
SimmondsP.1997; Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by Quantiplex HCV RNA assay versions 1 and 2, Roche monitor assay, and an in-house limiting dilution method. Journal of Clinical Microbiology 35:187–192
JarvisL. M.,
DavidsonF.,
HanleyJ. P.,
YapP. L.,
LudlamC. A.,
SimmondsP.1996; Infection with hepatitis G virus among recipients of plasma products. Lancet 348:1352–1355
LanfordR. E.,
SureauC.,
JacobJ. R.,
WhiteR.,
FuerstT. R.1994; Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 202:606–614
LanfordR. E.,
ChavezD.,
VonchisariF.,
SureauC.1995; Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rtth reverse transcriptase PCR. Journal of Virology 69:8079–8083
LaskusT.,
RadkowskiM.,
WangL.-F.,
VargasH.,
RakelaJ.1997; Lack of evidence for hepatitis G virus replication in the livers of patients co-infected with hepatitis C and G viruses. Journal of Virology 71:7804–7806
LearyT. P.,
MuerhoffA. S.,
SimonsJ. N.,
PilotmatiasT. J.,
ErkerJ. C.,
ChalmersM. L.,
SchlauderG. G.,
DawsonG. J.,
DesaiS. M.,
MushahwarI. K.1996; Sequence and genomic organization of GBV-C: a novel member of the Flaviviridae associated with human non-A-E hepatitis. Journal of Medical Virology 48:60–67
LeratH.,
BerbyF.,
TrabaudM. A.,
VidalinO.,
MajorM.,
TrepoC.,
InchauspeG.1996; Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells. Journal of Clinical Investigation 97:845–851
McGuinnessP. H.,
BishopG. A.,
McCaughanG. W.,
TrowbridgeR.,
GowansE. J.1994; False detection of negative-strand hepatitis C virus RNA. Lancet 343:551–552
MasukoK.,
MitsuiT.,
IwanoK.,
YamazakiC.,
OkudaK.,
MeguroT.,
MurayamaN.,
InoueT.,
TsudaF.,
OkamotoH.,
MiyakawaY.,
MayumiM.1996; Infection with hepatitis Gb virus C in patients on maintenance haemodialysis. New England Journal of Medicine 334:1485–1490
MellorJ.,
HolmesE. C.,
JarvisL. M.,
YapP. L.,
SimmondsP.
The International HCV Collaborative Study Group 1995; Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions : implications for virus classification. Journal of General Virology 76:2493–2507
MoldvayJ.,
DenyP.,
PolS.,
BrechotC.,
LamasE.1994; Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization. Blood 83:269–273
MullerH. M.,
PfaffE.,
GoeserT.,
KallinowskiB.,
SolbachC.,
TheilmannL.1993; Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication. Journal of General Virology 74:669–676
NavasS.,
CastilloI.,
BartolomeJ.,
MarriottE.,
HerreroM.,
CarrenoV.1994; Positive and negative hepatitis C virus RNA strands in serum, liver and peripheral blood mononuclear cells in anti-HCV patients: relation with the liver lesion. Journal of Hepatology 21:182–186
O’DohertyU.,
SteinmanR. M.,
PengM.,
CameronP. U.,
GezelterS.,
KopeloffI.,
SwiggardW. J.,
PopeM.,
BhardwajN.1993; Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. Journal of Experimental Medicine 178:1067–1076
OuC. Y.,
CiesielskiC. A.,
MyersG.,
BandeaC. I.,
LuoC. C.,
KorberB. T.,
MullinsJ. I.,
SchochetmanG.,
BerkelmanR. L.,
EconomouA. N.1992; Molecular epidemiology of HIV transmission in a dental practice. Science 256:1165–1171
OunanianA.,
GueddahN.,
RolachonA.,
TheluM. A.,
ZarskiJ. P.,
SeigneurinJ. M.1995; Hepatitis C virus RNA in plasma and blood mononuclear cells in patients with chronic hepatitis C treated with alpha-interferon. Journal of Medical Virology 45:141–145
ShindoM.,
DibisceglieA. M.,
AkatsukaT.,
FongT. L.,
ScaglioneL.,
DonetsM.,
HoofnagleJ. H.,
FeinstoneS. M.1994; The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C. Proceedings of the National Academy of Sciences, USA 91:8719–8723
SimmondsP.,
BalfeP.,
PeuthererJ. F.,
LudlamC. A.,
BishopJ. O.,
BrownA. J.1990a; Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. Journal of Virology 64:864–872
SimmondsP.,
McOmishF.,
YapP. L.,
ChanS.-W.,
LinC. K.,
DusheikoG.,
SaeedA. A.,
HolmesE. C.1993; Sequence variability in the 5′ non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. Journal of General Virology 74:661–668
WangJ. T.,
TsaiF. C.,
LeeC. Z.,
ChenP. J.,
SheuJ. C.,
WangT. H.,
ChenD. S.1996; A prospective study of transfusion transmitted GB virus C infection: similar frequency but different clinical presentation compared with hepatitis C virus. Blood 88:1881–1886
WillemsM.,
PeerlinckK.,
MoshageH.,
DeleuI.,
VandeneyndeC.,
VermylenJ.,
YapS. H.1994; Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C -evidence for viral replication in peripheral blood mononuclear cells. Journal of Medical Virology 42:272–278
ZhangL. Q.,
SimmondsP.,
LudlamC. A.,
Leigh BrownA. J.1991; Detection, quantitation and sequencing of HIV-1 virus from the plasma of seropositive individuals and from factor VIII concentrates. AIDS 5:675–681