Figure 2. TBEV burden in central nervous system (CNS) of mice. A–E) Five 6–10-week-old female C57BL/6 mice were infected subcutaneously with 104 FFU of Torö-2003 (blue) or HB171/11 (red), and viral burden in CNS tissue (olfactory bulb [A], cerebrum [B], cerebellum [C], brain stem [D], and spinal cord [E]) was measured by quantitative PCR and normalized to intracellular glyceraldehyde 3-phosphate dehydrogenase levels. Horizontal black bars indicate mean values. Statistical significance calculated by the Mann-Whitney test (*p<0.05; **p<0.005). F) Survival analysis of C57BL/6 mice after intracranial inoculation with phosphate buffered saline (mock, black) or with 100 FFU of Torö-2003 (n = 8) or HB171/11 (n = 10) in 20 μL phosphate buffered saline. For intracranial infections, mice were anesthetized by intraperitoneal injection with a mixture of ketamine (100 μg/g body weight) and xylazine (5 μg/g body weight). Survival differences were tested for statistical significance by log-rank test (****p<0.0005). G) Viral replication kinetics in primary cortical neurons. Primary cortical neurons were isolated as described previously (11). The neurons were infected with Torö-2003 or HB171/11 strain with 0.001 multiplicity of infection at day 7 postseeding, and viral growth was determined at indicated time points by focus forming assay, as previously described (15). Statistical significance was calculated using unpaired t test (*p<0.05). a.u., arbitrary units; FFU, focus-forming units; TBEV, tick-borne encephalitis virus.