GB virus B (GBV-B) is the most closely related virus to the hepatitis C virus (HCV) and is an attractive surrogate model system for HCV drug development efforts. Unfortunately, GBV-B can only be grown in the primary hepatocytes of certain non-human primates. We grew GBV-B in tamarins and marmosets and then used this virus in the absence and presence of lipofection reagents to try to infect 20 different cell lines including human primary hepatocytes and marmoset primary hepatocytes. GBV-B only replicated in marmoset primary hepatocytes. We isolated primary hepatocytes from GBV-B-positive and negative tamarins and marmosets and tried to immortalize the cells using SV40 large T-antigen or cell fusion. GBV-B stable cell lines were constructed in Huh7 and HepG2 cell lines, but there was no evidence for viral replication or a response to antiviral agents in these lines. Infectious full-length GBV-B RNA could be transfected into Vero, Huh7 and HepG2 at high efficiency, however there was no evidence for GBV-B replication or a response to antiviral agents. None of these approaches were successful and an in vitro model of GBV-B replication using immortalized cell lines was not produced. We hypothesize that these immortalized cell lines lack liver-specific factors that are required for GBV-B replication.
[Show abstract][Hide abstract] ABSTRACT: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro.
HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively.
The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells.
HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.
World Journal of Gastroenterology 09/2006; 12(30):4836-42. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of beta-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.
Journal of General Virology 08/2008; 89(Pt 8):1911-20. DOI:10.1099/vir.0.2008/001131-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The poor response to antiviral treatment of hepatitis C virus (HCV)-infected patients with genotype 1b has been associated with a higher prevalence of metabolic syndrome. However, the molecular link between these clinical entities is not clear. The goal of this study was to clarify the role of genotype 1b and 2 in the genetic expression of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate 1 (IRS-1).
We infected human hepatocellular carcinoma cell line (HepG2) cells with human HCV genotype 1b or 2 and measured the gene and protein expression of SOCS3 at various times. We also evaluated impairment in the insulin pathway by analysis of IRS-1 and phospho-AKT. For the control, we used HepG2 cell cultures treated with non-infectious serum. We also demonstrated the occurrence of HCV infection by the detection of both positive and negative strands in the cells and culture medium. To test infection of the HepG2 cells, we performed quantitative real-time polymerase chain reaction (qRT-PCR) of viral load at different time points. We analyzed the viral genotype in the pellet and supernatant.
At each time point, we found positive and negative strands in the infected cells, while in the medium we found positive, but no negative strands. We also detected the presence of the correct genotype in the medium. Two weeks following infection when the viral load was higher, we tested genotype 1b and 2 infected cells. SOCS3 gene expression was significantly higher in genotype 1b-infected cells (median 2.56; mean 2.82+/-0.59) compared with genotype 2 (median 1.34; mean 1.46+/-0.31) (p=0.04) and control cells (median 1.09; mean 1.02+/-0.11, p=0.02). There was no difference between cells exposed to genotype 2 and control cells. Conversely, IRS-1 was significantly lower in genotype 1b-infected cells (median 15.97; mean 15.45+/-0.67) compared with genotype 2-infected cells (median 16.45; mean 16.44+/-0.01, p=0.04). Statistically significant differences were seen when comparing the pAKT/AKT ratio in genotype 1b-infected cells (0.19+/-0.034) and not genotype 1b-infected (genotype 2-infected and non-infected) cells (0.253+/-0.004, p=0.03). This inverse regulation is compatible with interactions between the molecular expression of SOCS3, IRS-1 and phospho-AKT mediated by the genotype 1b virus.
Up-regulation of the SOCS3 gene might be one of the mechanisms governing non-response to therapy and expression of insulin resistance mediated via a direct mechanism at this level of genotype 1b HCV.
Clinical Chemistry and Laboratory Medicine 10/2009; 47(10):1217-25. DOI:10.1515/CCLM.2009.280 · 2.71 Impact Factor
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