Correlation of Severity of Human Tick-Borne Encephalitis Virus Disease and Pathogenicity in Mice

Abstract

We compared 2 tick-borne encephalitis virus strains isolated from 2 different foci that cause different symptoms in tick-borne encephalitis patients, from neurologic to mild gastrointestinal symptoms. We compared neuroinvasiveness, neurovirulence, and proinflammatory cytokine response in mice and found unique differences that contribute to our understanding of pathogenesis.

Keywords: Germany; Sweden; Tick-borne encephalitis virus; arboviral diseases; flavivirus; pathogenesis; tick-borne encephalitis; ticks; vector-borne infections; viruses.

Figure 1

Survival analysis and TBEV burden in peripheral organs of Torö-2003–infected and HB171/11-infected C57BL/6 mice. A) Survival analysis of ten 6–10-week-old female C57BL/6 mice after subcutaneous inoculation with phosphate-buffered saline (mock, black) or with 10⁴ focus forming units (FFU) of Torö-2003 (blue) or HB171/11 (red) in 100 μL phosphate-buffered saline. Survival differences were tested for statistical significance by log-rank test. B, C) Viral burdens in spleen (B) and lymph node (C) after subcutaneous infection of Torö-2003 or HB171/11 (10⁴ FFU, n = 5) were measured by quantitative PCR and normalized to intracellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels as previously described (14). Each data point represents an individual mouse. D–I) Cytokine response in spleen (D–F) and lymph node (G–I). Expression levels of GAPDH, TNF-α (D,G), IL-6 (E,H), and CXCL-10 (F,I) were determined by validated QuantiTect primer assays (QIAGEN, Hilden, Germany) and quantitative PCR from organs prepared in B and C. Signals of indicated mRNA were normalized to the GAPDH mRNA signal. Bars indicate mean values. Asterisks indicate statistical significance calculated by Mann-Whitney test (*p<0.05; **p<0.01). Horizontal black bars indicate mean values. a.u., arbitrary units; CXCL, CXC motif ligand; IL, interleukin; TBEV, tick-borne encephalitis virus; TNF, tumor necrosis factor.

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.