Mosquito Infection with Arboviruses

Mosquitoes were orally infected with DENV2 or DENV4 via artificial membrane feeding, as previously described [8 ,15 (link)]. In brief, C6/36 cells grown to 80% confluence were infected with DENV2, DENV4, or ZIKV at a multiplicity of infection (MOI) of 3.5 and incubated at 32°C and 5% CO₂ for 6 days. The infected cells were then harvested and lysed through 3 cycles of freezing and thawing (between dry ice and a 37°C water bath). CHIKV was amplified on Vero cells at an MOI of 0.01 and harvested approximately 36 h later. The propagation yielded virus titers of 10⁶ to 10⁷ PFU/ml. The viruses were then mixed 1:1 v/v with commercial human blood and supplemented with 10% human serum and 1 mM ATP. The bloodmeal was offered to mosquitoes via an artificial membrane feeding system. Each experiment was performed in at least two to three biological replicates, as indicated. Plaque assays for DENV2 were performed in the BHK cell line, while CHIKV and ZIKV were titrated on Vero cell monolayers, and plaques were visualized by staining with 1% crystal violet. TCID50 assays for DENV4 were performed in C6/36 cells and visualized using peroxidase immunostaining, with monoclonal antibody 4G2 (a flavivirus group-specific monoclonal antibody) [16 (link)] as the primary antibody and a goat anti-mouse horseradish peroxidase (HRP) conjugate as the secondary antibody. All procedures involving DENV and ZIKV infections were performed in a BSL2 environment, and procedures involving CHIKV infections were performed in a BSL3 environment.