Speed mill plus homogenizer

To measure the levels of toxins in the liver, equal amounts of liver tissues were homogenized in normal saline (0.1 g/mL) with a Speed-Mill PLUS homogenizer (Analytik Jena, Jena, Germany), and the supernatants were obtained by being centrifuged at 3500× g for 10 min. The levels of endotoxins in mouse serum and liver homogenates were measured by using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA), according to the manufacturer’s manual.

Equal amounts (around 0.2 g) of tissues and counted cells were homogenized in RIPA buffer (0.1 g/mL) (Beyotime, China) by using a Speed-Mill PLUS homogenizer (Analytik Jena, Jena, Germany). The homogenate was then centrifuged at 12,000 × rpm for 20 mins at 4 °C, and the supernatant proteins were collected. After the measurement of protein concentration, the proteins were boiled in 1× sodium dodecyl sulfate (SDS) sample buffer (100 μl for 1× 10⁶ cells) for 5 mins, and then equal amounts of protein (50 μg) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK). The membrane was then incubated with specific primary antibodies (Additional file 4: Table S2) and fluorescently conjugated secondary antibodies (Licor, USA), followed by the detection with the Odyssey system (TAITEC Co., Saitama, Japan).

One hundred mg of liver or feces from each mouse were homogenized in hexane (200 mg/mL) with a Speed-Mill PLUS homogenizer (Analytik Jena, Jena, Germany). The supernatant was obtained by centrifugation at 8000 rpm for 10 min at room temperature. Three hundred μL of supernatant were transferred to new tube and evaporated by centrifugal concentration equipment, the residue was then weighed.

Adult ticks were individually homogenized in 500 µL L-15 medium without additives using six steel beads and a SpeedMill PLUS homogenizer (Analytik Jena AG, Germany). For nymphs and larvae, 200 μL media and 10 ceramic beads were used. Homogenization was performed in two to three cycles for 2 min at a frequency of 30 pulses/sec. The suspension was cleared by centrifugation at 2500 rpm for 10 min at 4°C. Pools were generated by combining 100 µL supernatants of 10 homogenized ticks each according to species, life stage, and sampling site.
Viral RNA was extracted using the QIAamp Viral RNA Mini Kit following the manufacturer’s instructions. RNA was transcribed in cDNA using Superscript III reverse transcriptase and random hexamer primers (Invitrogen GmbH, Karlsruhe, Germany). Pools were screened by PCR using primers based on a fragment of the S-segment of CCHFV as described before (Bergeron et al., 2015 (link)). Subsequently, supernatants of homogenates of individual ticks composing the PCR-positive pools were tested by PCR as described above. PCR products were sequenced by Seqlab (Göttingen, Germany). Sequences were analysed using Geneious Pro v9 (https://www.geneious.com) and compared to other sequences using the NCBI Basic Local Alignment Tool (Altschul et al., 1990 (link)).