Until today, the only reliable model for the study of HCV infection was the chimpanzee. Indeed, there was no robust in vitro infection system, yet. There was thus an urgent need for such an in vitro infection system in order to evaluate therapeutic agents.

Figure. Model for the infection of eukaryotic cells with hepatitis C virus in vitro. Removal of cell-bound lipoproteins from the low density lipoprotein (LDL) receptor might be the crucial step for efficient hepatitis C virus infection in vitro. a, the binding of the HCV-lipoprotein complex to the LDL receptor is hampered in vitro by the cell-bound lipoproteins and by the vast excess of free lipoproteins present in the human blood. b, prior to HCV infection, the cell-bound lipoproteins are removed from the LDL receptor by using dextran sulfate, thus generating free LDL receptors. c, lipoprotein-free LDL receptors can bind the HCV-lipoprotein complex, thus allowing adsorption and penetration of HCV into target cells. A similar involvement of lipoproteins might take place during the infection of cells with HBV.

Indeed, the current "state-of-the-art" model for the replication of HCV in tissue culture was based on the use of established hepatocyte cell lines, such as the HepG2 and the Huh-7 cell lines. The method of choice did employ the transfection of in vitro transcribed hepatitis C virus RNA replicons in the hepatoma cell line Huh-7. However, this progressively more efficient transfection procedure does not represent by any means an infection model. Although replicon-based assays do provide an in vitro subgenomic replication system which is precious for the screening of antiviral molecules, it is not relevant for the study of the early steps of infection and the evaluation of adsorption and internalization for the sake of receptor evaluation, and finally for neutralizations studies and prophylaxis.

It might be possible to inhibit the HCV infection in vivo with modified dextran sulphate molecules, as follows:

Figure. Model for the inhibition of hepatitis C virus infection in vivo. Therapeutic, modified dextran sulfate molecules in the blood should preferentially bind to the hepatitis virus-lipoprotein complex, but not to free lipoproteins (a). Upon removal of the cell bound lipoproteins by the hepatitis C virus-lipoprotein complex, if any, the free lipoproteins can bind to the LDL receptor. Binding of the hepatitis virus-lipoprotein complex to the LDL receptor is thus inhibited (b).)

The infection procedure described in this article might also allow the fine characterization of the viral infection at the cellular and molecular levels. Time-course studies for the detection of the nucleic acids and the viral polypeptides during the course of the HCV infection in vitro can be performed. An in vitro infection procedure might thus undoubtly reduce and spare the use of animals and especially chimpanzees for viral infection studies and for the screening of antiviral compounds. The relevance of an adequate in vitro infection system for the agent of hepatitis C virus, which constitutes a global health problem, deserves special emphasis.

It is crucial to emphasize the fact that in order to efficiently amplify the newly synthesized HCV RNA in the infected cells, the traces of dextran sulfate that are employed to remove the cell bound lipoproteins prior to the addition of the viral inoculum onto the cells have to be TOTALLY absent. If it is not the case, the remaining traces of dextran sulfate might inhibit the RT-PCR reaction. Therefore, after removal of the cell-bound lipoproteins, extensive washing of the cells with PBS must be performed; for example, six consecutive washes of 4 ml each for each well of a 6-well plate.