Development of Novel Small Molecule Antivirals Promoting Endocytosis Pathway Inhibition

1. Professor Bente and Dr. Cajimat will prepare the stocks of the live viruses to be used in these experiments . 2. The plaque reduction assays will be performed as follow: Vero E6 cells will be seeded in 96-well or 6-well tissue culture plates, then treated with different doses of each inhibitor (0-10 µM} for 1 hour and inoculated with the tested live virus strain (ANDV strain Chile-9717869), in the presence of the compounds. After adsorption of the virus for 1 hat 37°C, the virus-inhibitor mixture will be replaced with 100 µl or 3 ml of 0.9% methylcellulose in minimal essential medium (MEM) containing 10% fetal bovine serum and anti biotics, followed by incubation at 37 °C for 6 to 7 days for ANDV/SEOV and 10 to 11 days for SNV/PUUV. Plates will be fixed with 10% buffered formalin (ThermoFisher) before removal from the biocontainment laboratories, 3. Plaques will be imaged and counted, and the ICS0s will be calculated using the GraphPad Prism software, 4. The same sequence will be followed to test the live virus strain SNV (Sin Nombre ort ho-hant avirus, strain SN 77734), The same sequence will be followed to test the live virus strain PUUV (Puumala ortho-hantavirus}, The same sequence will be followed to test the live virus strain SEOV (Seoul ortho­ hantavirus).