EBLV-1 Infection of Bat Cells

The experiments were carried out using a previously established M. myotis cell line [40 (link)] derived from the olfactory nerve (MmNOl cells). The bat cell cultures were cultivated in $\text{Gibco}$ DMEM, High Glucose, $\text{GlutaMAX}$ medium (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS) supplement (Thermo Fisher Scientific) at 37  ${}^{\circ }$ C with 5% CO ${}_2$ . The cell-adapted EBLV-1 strain 8918FRA [64 (link)] was used, and virus stock was prepared on BSR cells.
The cells were cultivated in two groups; at 37  ${}^{\circ }$ C, to simulate euthermia and at 5  ${}^{\circ }$ C to simulate torpor. Each of the temperature groups contained an infected sample and a non-infected control (Fig. 1). The experimental design thus consisted of cells under four experimental conditions. All of them were inoculated in independent laboratory session triplicates to prevent technical errors in the results.
An amount of $2.5\times {10}^6$ cells was seeded on T25 flask. At 18 h after seeding, the cells were infected with lyssavirus EBLV-1 8918FRA at multiplicity of infection (MOI) 1 (i.e., $2.5\times {10}^6$ fluorescent focus-forming unit (FFU) in 700  $\mu$ L of medium without FBS. After 1 h, the cell culture medium was replaced with a fresh, virus-free medium containing 10% FBS and the cells were incubated for 6 h at 37  ${}^{\circ }$ C with 5% CO ${}_2$ . Then, the cells were incubated 48 h at 37  ${}^{\circ }$ C with 5% CO ${}_2$ or at 5  ${}^{\circ }$ C after closing the flasks.