Virus extraction mini kit

Sandfly pools were processed as previously described [22 (link)] in a final volume of 600μL, of which 200μL were used for total nucleic acid extraction using the Virus Extraction Mini Kit the BioRobot EZ1-XL Advanced (both from Qiagen). Elution was performed in 90μL of extraction buffer of which 5μL were used for RT-PCR and nested-PCR assays using primers targeting the polymerase gene and the nucleoprotein gene as previously described [20 (link), 23 (link), 24 (link)]. PCR products of the expected size were column-purified (Amicon Ultra Centrifugal filters, Millipore) and directly sequenced.

All samples of various developmental stages (larvae, pupae, and adults) were processed at the Unité des Virus Emergents in Marseille. Before processing, L1 and L4 were washed in physiological solution to remove larval feces and particles of infectious food. Each sample was homogenized individually in 600 μL of Eagle minimal essential medium (EMEM) supplemented with 7% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine (200 mM) using a Mixer Mill MM300 (Qiagen, Courtaboeuf, France) in the presence of a 3-mm tungsten bead. The resulting homogenate was centrifugated at 5000 g for 5 min to separate supernatant. 200 μL of supernatant was processed further and rest was stored at −80 °C. Viral nucleic acid was extracted by the Virus Extraction minikit (Qiagen) by BioRobot EZ1-XL Advanced (Qiagen) and eluted into 90 μL. Five microliters of this solution was used for real-time RT-PCR performed by SuperScript® III Platinum® One-Step qRT-PCR Kit w/ROX (Invitrogen, Villebon sur Yvette, France) according to manufacturer’s protocol on a CFX96 real-time system (Bio-Rad, Marnes la Coquette, France): (i) 48 °C for 30 min, (ii) 95 °C for 2 min, (iii) 95 °C for 30s, (iv) 60 °C for 1 min; steps (iii) and (iv) were repeated 45x. Primers and probes designed for the nucleoprotein gene specific for MASV were described previously [14 (link)].