CORE 6: IN VITRO VIROLOGY SUMMARY QCRG Pandemic Response Program In Vitro Virology Core leverages in-depth virology expertise from 10 groups to support Projects 1–6 in discovering and developing antivirals against coronaviruses and other RNA viruses with pandemic potential. The goal is to identify lead compounds from hits using live-virus assays and channel them, in an iterative process, through lead optimization to obtain Optimized Leads for each project. The In Vitro Virology Core is led by Melanie Ott (Gladstone, UCSF) and supported by co-Is Adolfo Garcia-Sastre, Ana Sesma (Icahn School of Medicine at Mt Sinai), Greg Towers, Clare Jolly (University College London), Luis Martinez-Sobrido (UT San Antonio), Marco Vignuzzi, Carla Saleh (Institut Pasteur), and Lorena Zuliani- Alvarez (UCSF). We will provide live virus, reverse genetics and subgenomic virological assays in cell lines and advanced primary cell models for 20 RNA viruses, including various coronaviruses, picornaviruses, togaviruses, flaviviruses, paramyxoviruses and bunyavirales. We will also provide tight organizational oversight with precisely defined and safe work- and data flows, central interpretation of results, and guidance on pan-antiviral potential of lead compounds. In Vitro Virology Core members will meet monthly and interface tightly with all projects and cores. Aim 1 will test inhibitors against coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2, hCoV-OC43, NL63, 229E and HKU1) in a tiered approach, initially using nanoluciferase SARS-CoV-2 reporter virus assays and later parallel, multi-site IC50, IC90 and CC50 determinations with various SARS-CoV-2 isolates. Compounds will be selected for further evaluation of their: (a) pan-coronavirus inhibition, (b) efficacy in lung organoids and primary lung epithelial cells grown at the air-liquid interface, (c) mechanism of action (with the Proteomics Core), and (d) combination studies including polymerase inhibitors remdesivir and molnupiravir. Resulting leads will be serially passaged in cell lines to identify drug-resistance mutations that will be cloned into SARS-CoV-2 molecular clones and studied for lead optimization. We will also provide reagents and pre-formed virus-like particles to Projects 3 and 6 for hit identification. Aim 2 will test inhibitors against other RNA viruses with pandemic potential, including enteroviruses EV-A71 and EV-D68, chikungunya virus, coxsackie viruses, poliovirus, rhinovirus, zika and dengue viruses and paramyxovirus, in live virus assays using viral isolates and engineered molecular clones. In a tiered approach similar to Aim 1, Target Characterization, Hit-to-Lead, and Lead Optimization steps will be performed in appropriate cell lines, as well as in lung and gut organoids and tonsil histocultures. Combination and resistance studies will be performed as described above and will include the pan-serotype dengue inhibitor JNJ-A07. If appropriate, we will test lead compounds from Aim 1 for antiviral act...