2.3. Antiviral Assay against HBV Genotype B in Huh7 Cells

Huh7 cells were seeded in T-75 cell culture flask using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. Cells were transfected with 6.75 μg pHBV 1.3-B6.2 (kind gift from Dr Lih-Hwa Hwang, National Yang-Ming University, Taipei, Taiwan) plasmid, using Lipofectamine 2000 reagent (ThermoFisher, Waltham, MA) according to the manufacturer's protocol. Four hours after transfection, cells were washed with PBS, trypsinized, and seeded in 96-well plate at 20,000 cells/well density, in the presence of serial dilutions of SRI-32007. Six days post-treatment, viability of the cells was measured by MTS assay as described previously for HepG2.2.2.5 cells. HBe antigen levels in the cell culture supernatant was measured by HBe antigen ELISA (Cat#WB2496, Xpressbio, Frederick, MD) using the kit protocol with slight modification. Briefly, 25 μL of cell culture supernatant was transferred to each well of ELISA plate in the presence of 25 μL of PBS with 50 μL of HRP-conjugate and incubated for 60 minutes at 37°C. ELISA plate was washed five times with ELISA washing buffer, three times with PBS followed by addition of 100 μL of QuantaRed Substrate (enhanced chemifluorescent substrate) working solution (Cat#15159, Thermo Fisher Scientific, Waltham, MA), and incubated for 15 minutes. Ten microliter of QuantaRed stop solution was added to each well, followed by shaking for 30 seconds and reading in the Spectramax I3 ELISA plate reader (Molecular Devices) (top read) at the 530 nm excitation and 585 nm emission wavelengths. HBe Ag levels in the compound-treated wells were normalized to the virus-infected wells without any compound treatment (virus control, VC). EC₅₀ was calculated as the compound concentration at which the HBe antigen secretion was reduced by 50% compared to the virus control (VC).