Viral Kinetics in Mosquitoes

In order to determine if the timing of the infectious blood meal affected the within-mosquito viral kinetics, mosquitoes were sampled at 5, 8 and 11 days post-infection (dpi) to test for infection and dissemination across all groups (sample sizes are provided in Additional file 1: Table S1). Mosquito legs and bodies were placed into separate tubes containing 900 μl BA-1 diluent media and BBs. Samples were then homogenized twice at 25 Hz for 3 min using a Qiagen Tissuelyzer (Qiagen, Hilden, Germany). RNA was extracted (5× MagMax 96 Viral RNA Isolation Kit; Applied Biosystems, Foster City, CA, USA) and tested for the presence of viral RNA via qRT-PCR (SuperScript III One-Step RT-PCR System; Invitrogen, Carlsbad, CA, USA) as in [40 (link)]. Each treatment was repeated a total of three times and data was averaged over these replicates. Differences among the treatment groups for infection and dissemination rates were tested by a chi-square test for multiple proportions on 5, 8 and 11 dpi.