Molecular screening for tick-borne encephalitis virus in ticks from urban environments

Climate change has significantly influenced tick ecology by extending their seasonal activity and facilitating their expansion into new habitats. As a result, the incidence of tick-borne diseases is rising in previously low-risk areas, posing increasing public health concerns. Among these, tick-borne encephalitis (TBE) is one of the most significant vector-borne diseases in Europe, with the Baltic countries accounting for nearly one-third of reported European cases. This study assessed the prevalence of tick-borne encephalitis virus (TBEV) in questing ticks in Lithuania and key risk factors for transmission. Between 2023 and 2024, 544 ticks (Ixodes ricinus, n = 525; Dermacentor reticulatus, n = 19) were collected using the flagging method in urban areas and grouped into 47 pools based on species and developmental stages. TBEV detection was performed using real-time RT-PCR and virus isolation in two cell lines (Neuro-2a, MARC-145). TBEV RNA was detected in 15.26% (83/544) of samples. Virus isolation proved more effective than direct RT-PCR, with a total minimum infection rate (MIR) of 4.41% across both cell lines, compared to 1.47% from direct RT-PCR. The MIR for I. ricinus was 1.3% (7/525) and for D. reticulatus 5.3% (1/19). Positive samples included six nymphs, nine females, and seven males, highlighting widespread TBEV circulation across developmental stages and sexes. These findings suggest that D. reticulatus may play a greater role in TBEV transmission than previously recognised. The study underscores the need for ongoing tick surveillance and public health strategies, as well as the importance of virus isolation for improving detection accuracy.