Fluorescence microscopy

BHK-21 cells (in MAYV, SINV and VSV assays) or Vero cells (in ZIKV^BR, ZIKV⁷⁶⁶ and CHIKV assays) seeded in 24-well plates were infected with either CoPPIX-, SnPPIX- and heme-treated or non-treated viral particles for 1 h at 37 °C and 5% CO₂. After that, medium was replaced by fresh medium containing 2% FBS. After 20 h (for MAYV, SINV, VSV and CHIKV) or 48 h (for ZIKV^BR and ZIKV⁷⁶⁶), the plates were fixed with 4% formaldehyde in PBS for 15 min. The production of viral proteins was assessed using either an anti-alphavirus E protein mouse monoclonal antibody (in analyses with MAYV, SINV and CHIKV) or the conditioned medium of 4G2 hybridome (anti-flavivirus mouse antibody, used to mark ZIKV envelope protein), both coupled with a goat anti-mouse Alexa Fluor 488 (Invitrogen). Images were taken on an inverted fluorescence microscope with magnifications of 10 and 40x.