Three- to five-day old female mosquitoes were starved for 24 h and fed on an infectious blood meal containing 40% volume of washed erythrocytes from specific pathogen free (SPF) pig’s blood (Prestige BioResearch, Singapore), 5% 10 mM ATP (Sigma-Aldrich, USA), 5% human serum (Corning human AB serum, USA) and 50% virus solution in RPMI media (Gibco, USA), using Hemotek membrane feeder system (Discovery Workshops, UK). The virus titers in blood meals were 2 × 107 pfu/ml for DENV2, 6 × 106 pfu/ml for ZIKV, and 1.5 × 108 pfu/ml for CHIKV, which all resulted in 100% SG infection15 (link). Bloodmeal titers were validated by plaque assay using BHK-21 cells. Control mosquitoes were fed with the same blood meal composition except for the virus solution, which was replaced by RPMI media. Following oral feeding, fully engorged females were selected and kept in a cage with ad libitum access to a 10% sucrose solution in an incubation chamber with conditions similar to insect rearing.
For inoculation, mosquitoes were cold-anesthetized and intrathoracically injected with 0.5 pfu of either DENV2, ZIKV or CHIKV using a Nanoject-II (Drummond scientific company, USA). The same volume of RPMI media was injected as control. Virus inoculation was conducted four days post dsRNA injection.
For inoculation, mosquitoes were cold-anesthetized and intrathoracically injected with 0.5 pfu of either DENV2, ZIKV or CHIKV using a Nanoject-II (Drummond scientific company, USA). The same volume of RPMI media was injected as control. Virus inoculation was conducted four days post dsRNA injection.
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