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.