Mosquito Body and Leg Viral RNA Extraction

Individual mosquito bodies and legs were homogenized with metal beads for 4 min at 20 Hz (Mixer Mill Retsch MM301, Germany) using cell-culture medium supplemented at 20% FBS for bodies and NucliSENS lysis buffer (bioMérieux, France) for legs [19 (link)]. Homogenate supernatants were recovered after centrifugation 5 minutes at 20,000 x g. Nucleic acids were extracted with the NucliSENS miniMAG system (bioMérieux, France) in accordance with manufacturer’s instructions. Real time RT-PCR were performed on a CFX96 Touch Real-Time PCR Detection System instrument using iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories, France) and the primers and probes previously described [4 (link)].