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.