Mosquito Saliva Titers and CHIKV Detection

Twenty females were used every 1–2 days until day 14 pi. Females were chilled, and wings and legs were removed and discarded. Proboscis was inserted into 1 µL micropipette (microcaps®, Drummond Scientific Company, PA, USA) filled with 1 µL of Fetal Bovine Serum (FBS). One µL of 1% pilocarpine, an analogue of the acetylcholine, prepared in PBS at 0.1% Tween 80, was applied on the thorax to stimulate salivation. After 45 min, medium containing the saliva was expelled under pressure into 1.5 mL tubes containing 29 µL of DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS. These 30 µL were added, diluted or undiluted, to monolayers of Vero cells to detect infectious particles by the plaque assay technique. Cells were incubated for 3 days at 37°C under an overlay consisting of DMEM 2×, 2% FBS, antibiotics and 1% Indubiose (IBF Biotechnics). Plaques were counted after staining with a solution of crystal violet (0.2% in 10% formaldehyde and 20% ethanol). The titer of infectious particles per saliva was expressed as PFU/mL. One assay was achieved for each mosquito species.
In addition to collection and titration of saliva, 5 females at different days pi were tested for the presence of CHIKV on head squashes to evaluate the relation between the presence of CHIKV in saliva and head squashes positive by IFA.