Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+.

BACKGROUND: Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin's lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3+, CD14+ and CD19+. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV. METHODS: PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3+, CD14+, CD19+ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining. RESULTS: Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3+ cells then in 8/26 (30.8%) of CD14+ and CD19+ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3+ (2.4%), then in CD19+ (1.2%), and much lower in CD14+ (0.4%) cells. CONCLUSIONS: Our results indicate that CD3+ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5' Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing.

Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5' untranslated region (5'UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5'UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS.

Genetic Variability of Hepatitis C Virus (HCV) 5' Untranslated Region in HIV/HCV Coinfected Patients Treated with Pegylated Interferon and Ribavirin.

Association between hepatitis C virus (HCV) quasispecies and treatment outcome among patients with chronic hepatitis C has been the subject of many studies. However, these studies focused mainly on viral variable regions (E1 and E2) and usually did not include human immunodeficiency virus (HIV)-positive patients. The aim of the present study was to analyze heterogeneity of the 5' untranslated region (5'UTR) in HCV/HIV coinfected patients treated with interferon and ribavirin. The HCV 5'UTR was amplified from serum and peripheral blood mononuclear cells (PBMC) samples in 37 HCV/HIV coinfected patients treated for chronic hepatitis C. Samples were collected right before treatment, and at 2, 4, 6, 8, 12, 20, 24, 36, 44, 48, 60, and 72 weeks. Heterogeneity of the 5'UTR was analyzed by single strand conformational polymorphism (SSCP), cloning and sequencing. Sustained virological response (SVR) was achieved in 46% of analyzed HCV/HIV co-infected patients. Stable SSCP band pattern was observed in 22 patients (62.9%) and SVR rate among these patients was 23%. Decline in the number of bands and/or shift in band positions were found in 6 patients (17.1%), 5 (83%) of whom achieved SVR (p=0.009). A novel viral genotype was identified in all but one of these patients. In 5 of these 6 patients a new genotype was dominant. 5'UTR heterogeneity may correlate with interferon and ribavirin treatment outcome. In the analyzed group of HCV/HIV coinfected patients, viral quasispecies stability during treatment favored viral persistence, whereas decrease in the number of variants and/or emergence of new variants was associated with SVR. Among injection drug users (IDU) patients, a new genotype may become dominant during treatment, probably due to the presence of mixed infections with various strains, which have different susceptibility to treatment.

Hepatitis C virus (HCV) infection of peripheral blood mononuclear cells in patients with type II cryoglobulinemia.

OBJECTIVES: Type II cryoglobulinemia is a common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. The mechanisms behind its development are unclear, but could be related to direct infection of the immune cells. METHODS: Peripheral blood mononuclear cells from 18 patients with type II cryoglobulinemia were separated into CD3+ (T cells), CD19+ (B cells) and CD14+ (monocytes) and analyzed for the presence of negative strand HCV RNA, which is a viral replicative intermediate, and for the presence of HCV non-structural protein 3 (NS3). Control group consisted of 182 consecutive chronic hepatitis C patients prior to initiation of antiviral therapy. RESULTS: Negative strand HCV RNA was detected in PBMC from six (33.3%), patients and in 15 (8.2%) controls (p < 0.01). Negative strand was most frequently detected in B cells (3 patients), followed by T cells (2 patients), and monocytes (2 patients). One patient was positive both in CD3+ and CD14+ cells. NS3 protein was detected in six (33.3%) patients; five were positive in T cells, three in B cells, and another three were positive in monocytes. Two patients were positive in all analyzed cell subpopulation and one patient was positive in CD14+ and CD19+ cells, but not in CD3+ cells. Altogether, 11 patients (61.1%) were positive either for the negative strand HCV RNA or NS3 protein in at least one of the analyzed cell compartments. CONCLUSION: Our findings of common presence of viral replication in cells of the immune system suggest that direct HCV infection could play a role in the etiology of cryoglobulinemia.

Hepatitis G virus/GBV-C in serum, peripheral blood mononuclear cells and bone marrow in patients with hematological malignancies.

BACKGROUND: HGV/GBV-C is highly prevalent in the general population but its significance remains unclear. It is known that HGV/GBV-C is not primary hepatotropic and its replication was reported in PBMC, bone marrow and other tissues. To investigate a possible role of HGV/GBV-C 115 consecutive patients with hematological malignancies were analyzed for virus RNA presence and quasispecies composition in three compartments: serum, PBMC and bone marrow. METHODS: RT-PCR was used to amplify 5'UTR HGV/GBV-C in serum, PBMC and bone marrow. Viral sequences obtained from three compartments were subjected for comparative molecular analysis performed by single strand conformational polymorphism (SSCP) and pyrosequencing. RESULTS: HGV/GBV-C RNA was detected in 23 out of 115 (20.0%) patients, most often in bone marrow (18 patients), followed by PBMC (11 patients) and serum (10 patients). Differences in SSCP bands distribution corresponding to different viral variants and confirmed by direct sequencing were observed in three patients. CONCLUSION: HGV/GBV-C infection is frequent in patients with hematological malignancies. Common detection of HGV/GBV-C in bone marrow supports the hypothesis that it is a major replication site of this virus.