Mosquito RNA/DNA Extraction Protocol

For RNA and DNA extraction, mosquito pools were processed as described by Scheuch et al. [40 (link)]. Briefly, samples were homogenized with three 3 mm steel beads in 450 µL (for single mosquitoes) or 750 µL (pools with more than one specimen) serum-free minimal essential medium, supplemented with penicillin, streptomycin, gentamicin and amphotericin B (ThermoFisher Scientific, MA, USA), for 2 min at 30 Hz in a Tissue Lyzer II (Qiagen, Hilden, Germany). Subsequently, debris was pelleted by centrifugation for 3 min at 20,000 g. 200 µL of the supernatant was heat-inactivated (10 min, 70 °C) before simultaneous DNA/RNA extraction according to the manufacturer’s instructions, using the NucleoMag Vet Kit (Macherey-Nagel, Düren, Germany) on a BioSprint 96 workstation (Qiagen). Extracted nucleic acids were used for RT-qPCR virus analysis, RT-PCR and sequencing, as well as for genetic mosquito species identification and biotype differentiation.