in vitro replication of hcv rna in peripheral blood mononuclear cells isolated from patients undergoing treatment for hepatitis c virus infection

Background: although the liver is the main site for the hepatitis c virus (hcv) replication, there is still an essential debate about extrahepatic hcv reservoirs. objectives: it has been proposed that peripheral blood mononuclear cells (pbmcs) could be the possible virus replication sites. therefore, pbmcs may be candidates for recurrent hcv infection after achieving sustained virologic response (svr). in this study, we designed a lymphocyte culture to explore more about virus replication in pbmcs collected from patients with chronic hepatitis c. methods: plasma and pbmc samples were collected from 16 randomly selected seropositive patients for the anti hcv antibody. four out of 16 (25%) patients received combination therapy with alpha interferon and ribavirin. pbmcs were isolated from whole blood. between 10^6 - 10^7 cells were cultured with optimized concentrations of il2 (10 mg/ml) and phytohemagglutinin a (5 mg/ml). total rna was extracted from the first collected sera and harvested lymphocytes. constructed plasmids containing the ncr coding region were used to plot the standard curve for the relative quantification of sybr green realtime pcr. the sensitivity and specificity of the detection were established by using plasmids containing cdna.[results]with this plasmid containing the ncr coding region, the limit of detection (lod) of inhousedeveloped realtime rtpcr sensitivity was 2×10¹ copies. using primers for the ncr region, 10 out of 16 (62.5 %) pbmcs were positive for negativestrand hcv rna. among the four samples collected from patients with svr, negativestrand hcv rna was found in two patient samples.[conclusions]our results indicated that cultured lymphoid cells from patients with chronic hepatitis, even with svr, in the presence of il2 and pha, markedly enhanced the detection of hcv rna replicative strands. therefore, pbmcs may be reservoirs for recurrent hepatitis infection after svr and antiviral treatment. however, more clinical samples and control groups (lymphocyte culture without mitogen) should be examined to support the data presented in this study.