Frozen mosquito body samples were homogenized in a Bullet Blender Storm (Next Advance), according to a previously reported protocol (Fros et al., 2015b (link)). In total, 30 μl of the body tissue homogenate or mosquito saliva was added to one well of a 96-well plate containing Vero cells at 80% confluency. After incubation for 2 h at 37°C, the cell medium was replaced with fresh DMEM HEPES complete medium, and the cells were kept at 37°C for another 6 days. Positive viral infection was determined by checking for CPE at both 3 dpi and 6 dpi for each well. The infection rate and transmission efficiency were expressed as the percentage of virus-positive mosquito bodies or saliva over the total number of mosquitoes used for the salivation assay. Viral titers were determined by EPDAs using six and four bodies and saliva samples of the USUV-NL and USUV-IT infected mosquitoes, respectively.
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