The project established a robust infection protocol for juvenile Ixodes ricinus ticks with tick‑borne encephalitis virus (TBEV) and evaluated its suitability for subsequent bank vole (Myodes glareolus) infection studies. Immersion of larvae in a 1 × 10^8 PFU/ml TBEV Neudörfl isolate at 32 °C for 45 minutes yielded a 66 % survival rate after a three‑day incubation. Of the surviving 74 larvae, 93 % tested positive for viral RNA by RT‑qPCR, with an average load of 5.9 × 10^3 copies per tick. For nymphs, a 50 % relative humidity pre‑incubation for 24 hours followed by immersion produced a 95 % survival rate. After 14 days, 90 % of the 41 tested nymphs were TBEV‑positive, with an infection rate of 90.23 %. Viral loads increased from 4 – 4.9 × 10^4 copies per tick at day 3 to 5 × 10^5 (no pre‑treatment), 7 × 10^6 (50 % humidity), and 9 × 10^6 copies (leg‑removed) at day 14, indicating active viral replication. Infectious virus titers measured by plaque assay ranged from 1 × 10^3 to 3.16 × 10^6 PFU/ml in pre‑treated nymphs. Mortality after 14 days was highest (50–74 %) in the leg‑removed group, whereas the 50 % humidity group showed a lower mortality of 20–25 %. These data demonstrate that immersion combined with a 50 % humidity pre‑treatment provides the optimal balance of high infection efficiency and tick survival.

Attempts to generate saliva‑shed virus from immersion‑infected ticks revealed only 6.8 RNA copies per tick in feeding chambers, despite an average of 4 × 10^6 copies per tick in the chamber, suggesting limited virus excretion via saliva and the need for further model refinement before animal transmission studies. Injection of nymphs was not pursued due to safety concerns in the Czech Republic and a high mortality (80 %) observed in a separate West Nile virus study. Consequently, immersion remains the preferred method for producing large numbers of infected ticks for future experiments.

The project was carried out from autumn 2020 until its conclusion in late 2022, with milestone 1 (validation of the infection protocol) completed in the second half of 2022. Milestones 2 and 3, involving the establishment of a bank vole infection model and a full experimental tick‑vole transmission system, were delayed and ultimately not achieved because of reduced tick activity and COVID‑19 restrictions. The work was conducted in collaboration with the TP7 consortium, the Institute of Parasitology led by Prof. Dr. Christina Strube, and the Friedrich-Loeffler-Institut (FLI). A modified artificial feeding system based on Kröber and Guerin (2007) was employed, and data were documented in laboratory notebooks and digitally archived on a secure network. The project received funding from the German Research Foundation (DFG) and the Federal Ministry of Education and Research (BMBF), and its findings have already informed a DFG early‑career researcher grant (2021) on the molecular interaction of TBEV with ticks. A side study of tick populations in Lower Saxony identified a novel tick‑associated virus with potential mammalian host range, underscoring the broader relevance of the research.