Zika Virus Vector Competence Assay

Adult female mosquitoes were challenged with a ZIKV-containing blood-meal at 5 days post-eclosion. ZIKV I-44 strain (Genbank: KX856011) was used for challenges, isolated from mosquitoes from Mexico in 2016 [1 (link),24 (link)]. Prior to its use in vector competence studies, the virus had been serially passaged four times in Vero cells. ZIKV was added to 90% confluent Vero cells (ATCC: CCL-81) at multiplicity of infection (MOI) 0.01 for 96–120 hours or until 70% cytopathic effect (CPE) was observed. Vero cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 7% fetal bovine serum (FBS). Virus-containing supernatant was harvested and used immediately for artificial feedings in a 1:1 ratio with defibrinated sheep blood (Colorado Serum Company, Denver, CO, USA) supplemented with 10mM ATP to stimulate feeding. For each mosquito carton, the blood-meal was supplied through a parafilm (Thermo Fisher Scientific, Waltham, MA, USA) membrane stretched over a glass feeder, with the glass feeder heated by a water-jacket to 37°C. Mosquitoes were provided the infectious blood-meal for up to 1 hour and anaesthetised on ice for selection of engorged females, which were then kept in cardboard cartons in a humidified chamber at 28°C and 80% humidity until sampled.