Ribavirin

HepaRG cells were differentiated in six-well plates and infected for 24 hours with an HEV inoculum (faecal sample or supernatant) diluted in growth medium to a final volume of 1 mL. The viral suspension was then removed and cells were washed three times in PBS before adding 2 mL of growth medium. Every 2 to 3 days, one-half (1 mL) of the culture medium was replaced with fresh growth medium and infection maintained for up to 382 days (passage 2). Cells were not passaged during the duration of the infection. Six consecutive passages of the virus were then carried out onto fresh HepaRG cultures using this protocol. Details of the inoculum, MOI and length of infection used at each passage are presented in Table 1. Supernatants from infected cells used as inoculum were centrifuged prior to infection to remove cell debris. For ribavirin treatment, cells were infected for 12 to 13 days before addition of ribavirin (R9644, Sigma-Aldrich, Saint-Louis, MO, USA) into the supernatant to final concentrations of 0, 10, 50, 100 or 200 μM. Every 2 to 3 days, one-half (1 mL) of the culture medium was replaced with fresh growth medium containing the corresponding ribavirin concentrations and the cells treated for 21 to 30 days. Cells were then maintained for a further 13 to 14 days with one-half (1 mL) of the culture medium replaced with fresh growth medium without ribavirin every 2 to 3 days.

During HCV infection, Huh7.5 cells were pretreated with ribavirin (Sigma, St. Louis, MO) at 2 h prior to infection. When cells were treated with guanosine (Sigma, St. Louis, MO), 200 μM guanosine was added simultaneously with ribavirin. Cells were infected with 15 pmol/liter HCV from cell culture medium, as quantified by HCV core antigen measurement, in 300 μl in 12-well plates. After 4 h, cells were washed three times with buffered NaCl and medium, or medium supplemented with ribavirin was added. Cells were analyzed at 72 h postinfection.

ChemDiv was the source of the Serine/Cysteine Protease (SCP) inhibitor library used in this study. ChemDiv built the library using a variety of computational programs to qualify a small molecule as a potential SCP ligand (reviewed in [23] (link)). Basically, their method applied a set of pre-selected descriptors for encoding the molecular structures, and a trained neural network to qualify the molecules as potential SCP ligands. The molecular requirements were profiled by using available databases of active SCP and non-SCP-active agents. The library was composed of 849 compounds in a 96-well plate format and was used in both primary and secondary screening assays. For subsequent mechanism of action studies, compounds were reordered (ChemDiv, USA) at least three different times and tested independently to ensure consistency across batches. Reference reagents included human interferon-α2a (IFN-α) (PBL Interferon Source, USA), ribavirin (Sigma), Brefeldin A (cat # B-7450, Life Technologies) and Nocodazole (cat # M1404, Sigma). All compounds from the ChemDiv protease inhibitor library and ribavirin were prepared in 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) at 8 mM stock solution and stored at −20°C. IFN-α was prepared in Dulbecco's phosphate-buffered saline (DPBS, Sigma-Aldrich, USA) containing 5% FBS.