Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

jose-carlos Valle-Casuso; Delphine Gaudaire; Lydie Martin-Faivre; anthony madeline; Patrick Dallemagne; Stephane Pronost; Helene Munier-Lehmann; Stephan Zientara; Pierre-Olivier Vidalain; Aymeric Hans

EAV is a cytopathic virus. In this study, we took advantage of this specificity to develop a microplate 119 assay for the identification of EAV inhibitors. As a cellular model, we selected the ED cell line that 120 originates from equine dermis and thus matches the host specificity of EAV. To setup our in vitro 121 assay, we first analyzed the proliferation of non-infected ED cells in 96-well plates. To determine the 122 number of viable cells in each culture well, we quantified ATP which parallels the number of 123 metabolically active, living cells. Assays were performed using a commercial reagent based on the 124 principle that firefly luciferase luminescence is proportional and dependent on ATP concentrations 125 (CellTiter-Glo Luminescent Cell Viability Assay; Promega). In non-infected cells, ATP levels increased 126 by 41% in the first 24 h as a consequence of cellular proliferation, but then plateaued at 48 h as cell 127 cultures reached confluence ( Figure 1A ). Same experiment was performed using cell cultures infected 128 with the Bucyrus strain of EAV at a multiplicity of infection (MOI) of 0.5. As shown in Figure 1A as the number of genome copies increased by more than 2 logs in the first 24 h, and by 3 logs at 48 h 136 post-infection ( Figure 1B ). Finally, we showed that infectious viral particles are also produced by ED 137 cells. Indeed, at least 1x10 5 PFU/ml were detected in supernatants of infected cultures 48 h.p.i. 138 ( Figure 1C ). Therefore, ED cells represent an excellent in vitro model for EAV infection, and 139 quantification of ATP levels in culture wells is a convenient way to measure cytopathic effects 140 associated to viral growth. 141 142