Tick-borne encephalitis (TBE) is an emerging vector-borne and food-borne disease caused by the TBE virus, a member of the Orthoflavivirus genus, Flaviviridae family (Elbaz et al., 2022; Kunze et al., 2022; Postler et al., 2023). This virus is distributed across the Euroasian continent, forming a “TBE belt” stretching from Western Europe to the easternmost regions of Asia (Kunze et al., 2022). TBEV is classified into five subtypes based on antigenic characteristics, disease severity, and geographic distribution: European (TBEV-Eu), Siberian (TBEV-Sib), Far Eastern (TBEV-Fe), Baikalian (TBEV-Bkl), and Himalayan (TBEV-Him; Dai et al., 2018).

Tick-borne encephalitis-European primarily infects humans through the bites of infected Ixodes ricinus tick and occasionally via the consumption of unpasteurized milk products contaminated with TBEV. This European subtype is prevalent in Western, Central, and Southeastern Europe and is generally less virulent compared to TBEV-Sib and TBEV-Fe. Consequently, it is associated with milder forms of central nervous system (CNS) infections, more favorable outcome, and fewer long-term complications in survivors. Nonetheless, TBE remains a significant public health concern, particularly in Central and North-eastern Europe, with the highest number of cases reported in the Baltic countries, Czechia, Switzerland, Slovakia, Slovenia, Austria, and Germany (Steffen, 2016; Kunze et al., 2022).

Following exposure to TBEV, individuals can develop subclinical, abortive, or clinical infections. It is estimated that the majority of TBEV-Eu infections are subclinical, while approximately 30% progress to the febrile stage (Stage I) with concurrent viremia. Among these, 70% will clear the virus and have an abortive infection, while the remaining 30% will experience CNS involvement and develop manifest TBE (Barp et al., 2020; Bogovič et al., 2022a,b).

Due to the short viremia period and non-specific disease signs during Stage I (i.e., most patients seek medical attention when neurological symptoms appear), the definitive diagnosis of TBE relies on indirect methods, and most commonly enzyme-linked immunosorbent assays (ELISAs; Kunze et al., 2022). In regions where both TBEV and West Nile virus (Orthoflavivirus nilense; WNV) infections occur, such as Serbia, Hungary, Croatia, and Romania (Popescu et al., 2018; Zana et al., 2020; Petrović et al., 2021), ELISA results may lead to misdiagnosis of TBE as West Nile encephalitis and vice versa due to antibody cross-reactivity. Therefore, neutralization assays are often necessary for accurate differentiation (Potkonjak et al., 2017; Kunze et al., 2022).

Despite TBE being a notifiable disease in Serbia since 2004, there is limited awareness of the disease among medical practitioners (Vasić et al., 2022). The most recent detection of TBEV in ticks in Serbia dates back to 2017 (Potkonjak et al., 2017). Additionally, TBEV-reactive antibodies have been recently detected in individuals recovering from viral meningitis/encephalitis of unknown origin and in tick-infested patients (Banović et al., 2021, 2022a). Until recently, there was no active TBE surveillance program focused on serological or molecular screening of patients with CNS infection symptoms, and the assay for detecting TBEV-neutralizing antibodies (TBEV-NAb) was not available (Popović Dragonjić et al., 2022).

To assess the extent of TBEV exposure in the Serbian population and identify risk groups and high-incidence areas, we conducted a prospective observational study between 2019 and 2021. This study aimed to determine the prevalence of TBEV-NAbs in individuals who had been infested with ticks.

Tick-borne encephalitis virus foci are intricate microecosystems heavily influenced by a multitude of biotic and abiotic factors that can either facilitate or hinder virus transmission and the amplification chain (Cabezas-Cruz and Banović, 2023). The European continent is currently witnessing the expansion of TBEV endemic regions, primarily driven by climate changes, various anthropogenic factors, and the resulting migration of animals that serve as tick hosts (Kunze et al., 2022). Variation in these factors can result in disappearance of existing TBEV foci when the maintenance and transmission of the virus between vectors (i.e., Ixodidae) and reservoirs (e.g., small rodents of the genera Apodemus and Myodes) are disturbed (Knap and Avšič-Županc, 2015; Bournez et al., 2020). As TBEV foci expand, the incidence of TBE is on the rise across Europe, even in higher-altitude regions of Central Europe and previously considered TBEV-free areas such as Scandinavian countries (Kunze et al., 2022).

Despite TBE being a notifiable disease in Serbia, reported cases are infrequent due to low awareness among healthcare workers (Vasić et al., 2022). Another challenge in TBE diagnostics has been the absence of a gold standard diagnostic assay, as the detection of TBEV-neutralizing antibodies in Serbia only became possible in 2022 (Popović Dragonjić et al., 2022). Unlike many European countries where concerns over TBEV are growing, Serbia lacks an active surveillance program to assess the prevalence of TBEV infection in ticks and/or rodents. The last detection of TBEV in Serbia occurred in 2017 when infected I. ricinus ticks were collected in various locations within the suburbs of Belgrade and Fruška Gora Mountain (Potkonjak et al., 2017).

The existence of TBEV foci in different regions of Serbia was evident when tick-infested patients exhibited a higher likelihood of seroreactivity to TBEV antigens compared to blood donors from the same region (Banović et al., 2021). Further awareness arose when TBEV-reactive IgG were discovered in 20% of individuals recovering from viral encephalitis of unknown origin (Banović et al., 2022a; Cabezas-Cruz and Banović, 2023). However, a significant limitation of these studies is that TBEV exposure was inferred through assays not designed to differentiate anti-TBEV antibodies from the humoral response generated against other orthoflaviviruses circulating in Serbia (e.g., Orthoflavivirus usutuense and WNV). Indeed, when a neutralization assay was applied in a two-center seroprevalence study conducted in Novi Sad (Serbia) and Skopje (North Macedonia), no TBEV-neutralizing antibodies were found in the Serbian cohort exposed to tick bites (Jakimovski et al., 2023). This result may be influenced by the small number of participants included in the Serbian cohort (n = 51), compared with the cohort examined in this study (n = 450).

Among the 450 individuals exposed to tick bites, TBEV-neutralizing antibodies were found in three patients, representing a seroprevalence of 0.66%. This seroprevalence is considerably lower than the prevalence of IgG reactivity to TBEV in tick-infested individuals from Serbia (13.27%; Banović et al., 2021). This discrepancy is expected, as several members of genus Orthoflavivirus are circulating within Serbian population and causing development of specific immunoglobulins (Petrović et al., 2019, 2021) that can cross-react with other members of the Orthoflavivirus genus. In addition, TBEV-Nabs are only a subset of total anti-TBEV antibodies generated after exposure to TBEV. Although, TBEV-Nab seroprevalence in our cohort is higher compared to Norwegian, Romanian and Serbian blood donors (0.66 vs. 0.4% and 0.08 and 0%, respectively; Marvik et al., 2021; Coroian et al., 2022; Jakimovski et al., 2023) and much lower compared to residents of Czechia (0.66% vs. 26.3%; Kriz et al., 2015).

To identify potential TBEV foci in Serbia, we integrated anamnestic data and an epidemiological survey with scutal/coxal index values of ticks removed from patients with TBEV-neutralizing antibodies. We identified potential locations within three mountains: Fruška Gora Mountain, Divčibare Mountain, and Bukulja Mountain. Among these, Fruška Gora Mountain is the only locality previously suspected in seroprevalence and field studies to have an environment conducive to the maintenance of TBEV foci (Potkonjak et al., 2017; Banović et al., 2021). Regarding the tick species responsible for human infestations, our finding that I. ricinus was the most frequently removed tick aligns with previous studies conducted in Serbia (Banović et al., 2022b, 2023). Although I. ricinus ticks were removed from all three patients in whom TBEV-neutralizing antibodies were detected, we are not able to claim that TBEV exposure occurred during most recent infestation. Concerning the infestation time, the significance of age as a predictor level and the output of the Cox model used here necessitate further investigation. When a level of a predictor variable (age group) in a Cox proportional hazards model has a p value greater than 5%, it suggests that overall, that specific age group might not have a statistically significant difference in infestation time compared to other age groups. However, when conducting pairwise comparisons among levels of the predictor (i.e., age groups), it might be found that one particular age group significantly differs from another, even if its overall relationship in the main model was not statistically significant (Hosmer et al., 2011; Agresti, 2012).

A major limitation of this study is the absence of attempts to isolate and/or detect TBEV in ticks removed from humans, which could confirm recent exposure to the virus in patients with TBEV-neutralizing antibodies. Additionally, a 4-week period between tick infestation and serum sampling may not be long enough for all TBEV-exposed persons to develop detectable levels of TBEV-neutralizing antibodies. Uneven distribution of samples across the country limits the representativeness of the results for all districts of Serbia, necessitating further studies where several centers will be engaged in serum collection, where period between tick infestation and serum sampling should be expanded to at least 5 weeks. Nevertheless, this study is the first to provide unequivocal evidence of TBEV exposure in tick-infested individuals from Serbia. Since anti-TBEV IgM reactivity and anti-TBEV IgG avidity were not determined, we are not able to claim that most recent (i.e., observed) tick infestation is linked with virus exposure in patients with TBEV-neutralizing antibodies. Therefore, infestation locations should be considered as potential natural foci, warranting future surveillance campaigns. Healthcare institutions should initiate surveillance programs and test all cases of viral meningitis/encephalitis of unknown origin for the presence of TBEV-neutralizing antibodies, especially those responsible for the residents of Fruška Gora, Divčibare, and Bukulja mountains. This approach will enable the assessment of the TBE burden in the Serbian population, identification of risk groups, field research, and the implementation of prevention measures, including immunization and educational campaigns.

In this study, we have unveiled critical insights into the seroprevalence of TBEV-neutralizing antibodies and epidemiological significance of TBEV in Serbia. Although we were not able to provide the evidence of recent infection in tested patients, our findings suggest the existence of potential TBEV foci within Serbia, notably in regions proximal to Fruška Gora Mountain, Divčibare Mountain, and Bukulja Mountain. The detection of TBEV-neutralizing antibodies in tick-infested individuals, albeit at a relatively low prevalence of 0.66%, underscores the need for heightened surveillance efforts. The identification of TBEV exposure in these regions highlights the urgency of implementing active TBE surveillance programs. Furthermore, our study has shed light on the demographics of tick-infested individuals and the characteristics of tick infestations, including attachment times of different tick stages. Age was identified as a significant predictor of infestation time, emphasizing the importance of considering age-related factors in future TBE research and surveillance. Research efforts as presented in this paper are crucial for assessing the true burden of TBE, identifying high-risk groups, conducting further field research, and ultimately implementing effective prevention measures to mitigate the spread of this emerging disease.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

This study was approved by the ethical committee of Medical faculty Novi Sad, University of Novi Sad (Ethical approval No. 01-39/261/1 from 17.09.2020.) and conducted according to the Declaration of Helsinki and The Patient Rights Law of the Republic of Serbia. The studies involving humans were approved by Komisija za etičnost kliničkih ispitivanja Medicinskog fakulteta u Novom Sadu. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. The manuscript presents research on animals that do not require ethical approval for their study.

PB: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. DM: Formal analysis, Investigation, Methodology, Writing – original draft. IB: Conceptualization, Investigation, Methodology, Writing – original draft. VS: Conceptualization, Data curation, Investigation, Methodology, Writing – review & editing. EM: Formal analysis, Investigation, Methodology, Software, Visualization, Writing – review & editing. PK: Investigation, Methodology, Software, Supervision, Visualization, Writing – review & editing. KR: Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. NK: Investigation, Methodology, Validation, Writing – review & editing. MK: Data curation, Investigation, Resources, Validation, Writing – review & editing. TA-Ž: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – review & editing. AC-C: Data curation, Formal analysis, Methodology, Supervision, Writing – review & editing.