Rational development of tools to appropriately respond to SARSCoV- 2 needs

ABSTRACT

For many years, our laboratory has been developing cellular models for the study of human pathogenic viruses with RNA genomes, in order to study the replication of these pathogens, to propose new therapeutic pathways, to screen and test inhibitors. In response to the COVID-19 outbreak, we have set up the tools for the study of SARS-CoV-2 replication. First, clinical and reference SARS-CoV-2 strains have been successfully isolated and amplified using Vero E6 cells in the BSL3 facility of Bordeaux University (UB'L3, www.mfp.cnrs.fr/wp/larecherche/ andevir/ubl3/). We set up the monitoring of SARS-CoV-2 replication using conventional RT-qPCR quantification as well as evaluation of the cytopathic effect by microscopic observation or content analysis. Using VERO cells, we are now able to precisely titer viral supernatant (determination of the TCID50) and screen for potential antiviral molecule (determination of EC50 and CC50). We have developed a full-length Spike sequencing based on a Sanger approach1 as well as whole genome sequencing by nanopore technology, allowing the tracking of emerging variants. In parallel, we developed various other models to study SARS-CoV-2 replication including Calu-3 cells, modified human cells expressing Ace2 (e.g. 293T, U2OS) or even more complex cellular models (reconstituted human airway epithelium, vessels) according to the biological question to resolve. As an example, bronchial epithelia reconstituted from biopsies of adult or child donors were used to evaluate the inflammatory response upon SARS-CoV-2 infection in an age-dependent manner [2] (see poster G. Beucher). Similarly reconstituted blood vessels were used to study the impact of SARS-CoV- 2 infection on the vascular system and determine whether clinical observations (blood brain barrier damages, myocarditis) are due to direct infection of cells or indirect effects. Finally, we evaluate the efficacy of different chemical or physical processes for viral inactivation in air or on surfaces.