2.3. Virus Isolation

The suspensions of ticks, blood serum, and CSF samples were centrifuged at 12.000 g for 5 min and filtered with a 0.22 μm pore size microfilter (Techno Plastic Products AG, Trasadingen, Switzerland). Cell cultures of Vero (ATCC No. CCL-81) and Marc-145 (ATCC No. CRL-12231) were seeded in a maintenance medium containing Modified Eagle’s Medium (MEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) was used for murine neuroblastoma cells (Neuro-2a ATCC No. CCL-131) instead of MEM. All cells were incubated at 37 °C in 5% CO₂ overnight. Cells were inoculated with prepared tick suspensions or clinical samples from the dogs the following day. Cell cultures were examined for the occurrence of cytopathic effects through three serial passages which were performed in triplicates including triplicates of positive and negative controls for each round of analysis. After each serial passage, cell suspensions were harvested for RNA extraction. Successful virus isolation in cell cultures was confirmed using RT-nPCR. Partial genome sequencing targeting the NCR region of TBEV was used for internal confirmation of the specificity of detected TBEV RNA.