Zika virus stocks were used to inoculate Vero or C6/36 cells at a low (0.01 PFU/cell) or high (20 PFU/cell) MOI in 12-well plates. 3 or 5 days p.i. (for Vero or C6/36 cells, respectively) the cell culture supernatant was clarified by centrifugation and used for the next passage. After passage 1 (p1), blind passaging was performed, using 5 μl or 300 μl of the virus from the previous passage per well for low and high MOI conditions, respectively. All samples were titrated and we confirmed that indeed the high and low MOI conditions were respected (Supplementary Fig. 1). Yellow fever virus was passaged at high (5 FFU/cell) and low (0.1 FFU/cell) MOI in SW13 cells for 2 days. West Nile virus was passaged similarly, in C6/36 cells.

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