Study of the infection of human blood derived monocyte/macrophages with hepatitis C virus in vitro.

Hepatitis C virus (HCV) is essentially hepatotropic, but clinical observations based on quasispecies composition in different compartments or on viral RNA detection in cells suggest that the virus is able to infect and persist in cells other than liver cells. It was shown previously that peripheral blood mononuclear cells (PBMCs) are permissive to HCV replication in vitro but at a very low rate. Since different viruses associated with chronic infections are known to persist in monocyte/macrophages, it is important to determine whether these mononuclear blood cells are susceptible preferentially to HCV. In order to study HCV interaction with monocytes/macrophages, these cells were isolated from the blood of healthy donors and incubated with HCV genotype 1b positive sera. The detection by RT-PCR of the positive- and negative-strand RNA in the cells at different times and the increase in the amount of intracellular viral RNA measured by the branched DNA assay suggest that monocyte/macrophages can support HCV RNA replication. The rate, however, is very low. The analysis of hypervariable region 1 (HVR-1) nucleotide sequences indicated that some minor variant present in the inoculum might display a specific tropism for the monocytes/macrophages. These results provide evidence that human monocytes/macrophages might represent a reservoir for HCV. This cell tropism may be a crucial factor in the pathogenesis of hepatitis C.

Ultrastructural observations in hepatitis C virus-infected lymphoid cells.

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.

Sodium valproate, an anticonvulsant drug, stimulates human immunodeficiency virus type 1 replication independently of glutathione levels.

Since modulation of the glutathione (GSH) level has been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression, we have undertaken an analysis of the effect of sodium valproate (VPA) on HIV-1 replication. VPA, which is an anti-epileptic drug in widespread use in clinical medicine, has been shown to depress the activity of GSH reductase, an enzyme required for maintaining high cellular levels of reduced GSH. The effect of this drug on HIV-1 replication has been studied in primary infected cells, i.e. peripheral blood mononuclear cells (PBMC) and monocyte/macrophages, in the CEM-SS cell line, and in chronically infected stimulated and non-stimulated U1 cells. We have shown that VPA markedly enhanced viral replication in all infected cells tested. Virus production was induced in U1 cells by VPA treatment and the stimulatory effects of tumour necrosis factor-alpha, interleukin-6 and granulocyte/macrophage colony-stimulating factor were augmented. The LTR-driven gene expression in Jurkat T cells was increased. However, the elevated viral production did not correlate with the effect of VPA on the intracellular GSH level. Thus, VPA stimulated in vitro HIV-1 replication in acutely and chronically infected cells and enhanced LTR-driven gene expression. These effects were observed for concentrations that are reached in the plasma of VPA-treated patients. Therefore, although the clinical significance of these data remains to be demonstrated, these results should be considered in the choice of an anticonvulsant drug in HIV-infected individuals.