2.11 Virus titrations
Vero cells were grown to > 95% confluency in Dulbecco’s modified eagle media DMEM (5% fetal bovine serum (Atlas Biologicals), 1% sodium bicarbonate, 1% non-essential amino acids, no phenol red) in 6 or 12-well plates. Ten-fold serial dilutions of virus stocks and blood meal aliquots in media were performed. Mosquito samples were used undiluted. In vitro challenged mosquito samples had already undergone one freeze-thaw cycle prior to infectious virus detection. For each dilution or sample, one hundred microliters of sample was added to wells, then incubated with rocking for 1 hour, followed by an overlay (0.4% agarose (Lonza Rockland) in DMEM). At 2 days post-infection, overlays (0.33% neutral red (Sigma N2889), 2% agarose in supplemented DMEM) were applied. Plates were read after 24 hours. Ambiguous plaques were more closely examined under an inverted microscope at 40X magnification to better confirm CPE.