Collection of saliva and virus detection by plaque assay in Vero cells

Female mosquitoes at 7 and 14 dpi (Experiment 1) and at 7 dpi (Experiment 2) were immobilised on a Petri dish placed on ice and their legs and wings were removed with forceps. The proboscis of each live mosquito was inserted into a 10 μl pipet tip containing 5 µl of FBS and then expelled into a sterile 500 µl microcentrifuge tube with 45 µl of Leibovitz’s L15 medium supplemented with penicillin (10 μg/ml), gentamicin (1 μg/ml) and fungizone (1 μg/ml). After 30 min of salivation, saliva samples and mosquito bodies were immediately stored at -80 °C.

Vero cells were plated and incubated until monolayer formation. Then, the entire sample of the saliva (50 μl) for each mosquito was placed on a 6-well plate containing the cells and incubated for 1 h (37 °C, 5% CO₂) and supplemented with Earle’s 199 medium. After 1 h incubation, the supernatant was removed, and the cells were overlaid with carboxymethyl cellulose (CMC) in Earle’s 199 medium. Inoculated Vero cells were incubated for 7 days and then fixed with 10% formaldehyde and stained with crystal violet (0.02%). To ensure cell viability and to prevent self-contamination during the process, 50 μl of 3-fold serial diluted ZIKV stock in Leibovitz’s L15 medium was added in every assay to one of the plates as a positive control and cell culture medium alone as a negative control.