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)).
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 (
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