2.6 Vector competence
Adult mosquitoes (4-10 days old) were provided an oral, artificial meal containing freshly grown RVFV. To approximate titers of 7 log10 PFU/ml, virus was harvested 3 days after infection of Vero cells at an MOI of 0.01. Viral supernatant was mixed 1:1 in defibrinated calf blood, with the addition of 1mM ATP, and 0.075% sodium bicarbonate. Mosquitoes were fed for 1 to 1 ½ hours using either a water-jacketed feeder (https://lillieglassblowers.com) for DDVax and MP-12 or a hemotek (http://hemotek.co.uk/), in the case of ZH501. All ZH501 feedings and mosquito incubation steps were performed in the animal biosafety level 3 conditions. All other in vitromosquito oral exposures were conducted in standard biosafety level 3 containment. Fully-engorged mosquitoes were separated into cartons and provided sucrose and water ad libitum . Mosquitoes were held for 14 days at ~80% humidity and 28oC. Infectious blood meal titers were determined through back titration of the infecting blood meals.
At 14 days post-challenge, mosquitoes were anesthetized at 4oC, then held on ice during processing. Tissue samples were dissected, then placed in separate tubes of 250 µl mosquito diluent (DMEM, 20% heat-inactivated FBS, 50 µg/ml Pen-Strep, 50 µg/ml gentamicin, and 2.5 µg/ml amphotericin B), as follows: Legs and wings were removed for determination of viral dissemination. Saliva was collected for determination of transmission potential. The mosquito proboscis was placed in a capillary tube containing type B immersion oil (Bioworld, SKU- 21750002) and allowed to salivate for 30-60 minutes. At that time, the capillary tube was removed and placed in a tube containing mosquito diluent (1x PBS supplemented
with 20% FBS (heat-inactivated), 50 μg/ml Penicillin/Streptomycin, 50 μ/ml Gentamycin,
2.5μg/ml Fungizone) and centrifuged at 14,000 x g for 3 minutes. Lastly, each remaining body was also placed in a separate tube, for measurement of infection status. Samples were homogenized on a Qiagen Tissuelyzer (Qiagen) at 30 beats per second frequency for 30 seconds, then pelleted at 14,000 x g in a microfuge at 4oC for 3 minutes. Samples were stored in -80oC.