TBEV detection in questing and fed ticks
Only ticks collected from 2012 to 2014 were analysed. For questing ticks, adults were analysed individually and nymphs were analysed in a pool of one to five ticks. For fed ticks (collected on small mammals), larvae/nymphs were pooled per stage, engorgement status (fed, unfed), animals and month of capture with one to ten ticks per pool, and adults were analysed individually. Ticks were homogenised using 2.8 mm stainless steel beads in a Precellys 24 lyser/homogeniser (Bertin, France) at 5500 rpm for 20s. RNA was extracted using the Nucleospin RNA II kit (Macherey Nagel, Germany) according to the manufacturer’s instructions. Purified RNA was eluted into 50 µl RNase-free water and stored at -80°C until use. RNA samples were screened for TBEV by real-time RT-PCR targeting a 3′ non-coding region of the TBEV genome with specific primers and probes (Schwaiger & Cassinotti, 2003). Real-time RT-PCR Taqman assays were performed in a final volume of 20 µl using the LightCycler 480 RNA Master Hydrolysis Probes master mix (Roche Applied Science, Germany) according to the manufacturer’s instructions using 2 µl of RNA template. Positive and negative (water) controls were included in each run. Real-time RT-PCR thermal cycling conditions were as follows: 63°C for 3 min, 95°C for 30 s, 45 cycles at 95°C for 10 s, then 60°C for 30 s, followed by cooling at 40°C for 10 s. Conventional RT-PCR followed by nested PCR using primers targeting the non-structural protein gene NS5 (Puchhammer-Stöckl, Kunz, Mandl, & Heinz, 1995) were used to confirm the presence of TBEV in positive samples. Amplicons were sequenced by Eurofins MWG Operon (Germany), and then assembled using BioEdit software (Ibis Biosciences, Carlsbad). The online BLAST tool (National Center for Biotechnology Information) was used to compare results with published sequences listed in the GenBank sequence databases.