Isolation of tick-borne encephalitis virus from naturally infected dogs

Tick-borne encephalitis virus (TBEV) is an emerging concern in canine health [1–2], yet its active circulation in dogs remains insufficiently documented. In a cross-sectional study of 473 dogs from central Lithuania (2020–2021), we confirmed acute infections by PCR, serology, and virus isolation in cell culture. TBEV RNA was detected in 18.6% of canine samples, and antibodies were present in 21.6%. Importantly, we succeeded in isolating infectious TBEV from seven serum samples and one cerebrospinal fluid specimen. Replication was demonstrated in Neuro-2a, Vero, and Marc-145 cells, with subsequent confirmation by sequencing. These results provide direct evidence that viraemic dogs carry transmissible virus and can serve as a source for successful cultivation. In parallel, attached ticks collected from the same dogs carried TBEV RNA in 34.2% of suspensions. After three blind passages in cell culture, positivity rose to 56–61%, highlighting that viral replication in ticks often requires amplification to reach detectable levels. Nevertheless, no consistent concordance was found between a dog’s infection status and TBEV detection in its ticks. The study demonstrates that virus isolation from naturally infected dogs and their ticks is feasible and reliable, confirming the role of dogs as suitable sentinel hosts for TBEV. Cultivation in multiple cell lines expands opportunities for experimental studies and strain characterisation. Our findings underline the clinical and epidemiological importance of canine TBEV infections and provide valuable resources for further virological and pathogenesis research.