Influenza Virus Replication Assay

AX4/PB2 cells in MEM containing 0.3% (wt/vol) BSA were seeded on 384-well microplates (4,000 cells/well) unless otherwise stated, and various compounds were added to the cells (10% of the total assay volume) 18 h after seeding. One hour later, WSN/PB2-Rluc virus and L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin were added to the cells (20% of the total assay volume), and the assay plates were further incubated. The total assay volume was 10 µL/well, the multiplicity of infection (MOI) was 0.025, and the incubation time was 22 h, unless otherwise stated in the respective experiments. In the cell viability assay, viruses were not added to the cells. In the Rluc inhibition assay, compounds were added just before detection. Rluc activity was measured with the Renilla-Glo Luciferase Assay System (Promega). Virus contents in the cell culture supernatant were measured by means of plaque assays and with the NA-Star kit (Applied Biosystems). Cell viability was measured with the CellTiter-Glo assay system (Promega), according to the manufacturer’s instructions. Luciferase activity was measured with a microplate reader (EnVision; PerkinElmer). All dispensing processes were performed by using the Multidrop combi (Thermo) and the HORNET-NX (Wako).

For analysis of the spectrum of antiviral activity of the compound, the MDCK cell line served as the host cell. MDCK cells were seeded on 6-well microplates and were infected with various strains at an MOI of 0.0001. After a 1-h incubation, the infected cells were washed, and compound-containing medium was added to the cells. To examine virus replication kinetics, the cell culture supernatants were collected on days 1, 2, and 3 post-infection (pi). Virus titers in the supernatants were determined by use of plaque assays in MDCK cells.