Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.


Introduction
Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Europe and Asia.It is caused by the tick-borne encephalitis virus (TBEV, syn.Orthoflavivirus encephalitidis) (Flaviviridae: Orthoflavivirus) [1].TBEV can be divided into five subtypes, including the European, Siberian, Far Eastern, Baikalian, and Himalayan subtypes [2].In Europe, two of these are circulating-the Siberian subtype, causing Russian spring-summer encephalitis, which is transmitted mostly by Ixodes persulcatus, and the European subtype, which is widely spread over the western continent and mainly associated with I. ricinus and Dermacentor reticulatus ticks [2,3].
Unlike many other tick-borne pathogens (TBPs), which are widespread in the tick population, TBEV circulates in very strictly defined, patchy natural foci.The identification of these microfoci-small geographical areas in which TBEV was detected in questing ticks at least once-is, therefore, difficult and many such areas remain undiscovered to date.Usually, microfoci are found based on data provided by TBE patients as an indication of potential locations in which tick bites were acquired [4].Despite some limitations, the definition of risk areas, at least in Germany, helps formulate vaccination recommendations and monitor the spread of TBE.However, the TBE case incidence rate depends on temporal, climatic, and geographic factors, the vaccination coverage, the abundance of suitable tick vectors in the environment, and the population size of small mammals that are hosts of ticks and reservoirs for TBEV [5][6][7].However, exposure risk is a very significant factor in harboring tick-borne diseases.One well-known risk factor for a TBEV infection is spending over 10 h per week in forests [8].
The virus is transmitted to humans through tick bites, although the possibility of infection by consuming unpasteurized dairy products has also been described [9][10][11][12].In recent years, the TBE notification rate has increased in Europe, and the risk areas are expanding [13].In Germany, TBE is endemic in the southern part, with a more recent contiguous northeastward spread into Saxony and a patchy spread to northwestern regions [14,15].The number of reported cases in the last years varied from 400 to 700, and the federal states of Bavaria and Baden-Wuerttemberg account for more than 90% of them [4].In Poland, the annual rate of diagnosed TBE cases is between 200 and 300, with the majority of cases being reported in the north-eastern part of the country (Warmian-Masurian and Podlaskie Voivodships), which is the endemic area; however, an increasing number of cases have been also noted in western parts of Poland [8,16].
In previous research based on phylogeographic analyses [17,18], it had been demonstrated that tick-borne flaviviruses, in general, follow an east-to-west spreading pattern and that the TBEV strains isolated in Germany have derived from ancestor strains mainly originating in Austria and the Czech Republic [19].In our previous study, we isolated TBEV strains from northern Saxony, which showed very high similarity to isolates from Poland [20].Two of these isolates originated from areas not known to be TBE endemic.This suggests an alternative migration way for the virus in comparison to the one across the Bavarian Forest originating from the Czech Republic and may suggest that the geographic transmission of TBEV from Poland is faster than from the endemic south of Germany.
The main objectives of this study were to (i) identify new TBE microfoci and (ii) isolate and genetically characterize TBEV strains from ticks collected in yet nonendemic areas from states and voivodeships that are adjacent to each other across the national border to better understand the geographic spread of TBEV in these areas.

Tick Collection
Ticks were collected in areas that TBE patients had indicated as putative places for the tick bites acquired.To achieve this, patients were approached in two ways.Firstly, data from patients was gathered by notifying local health authorities in Germany and Poland.Secondly, online surveys were conducted.In both cases, patients were asked to complete an anonymous questionnaire (TBE diagnosis date, tick bite date, and potential geographic location of tick bite).Some patients were able to identify the exact area of the tick bite, while others pointed out multiple locations.In total, based on the geographical information provided by patients, 19 potential TBE microfoci from Northeastern Germany (states: Brandenburg, Mecklenburg-West Pomerania, Saxony) and Western Poland (voivodeships: Lower Silesia, Lubush) could be selected for further investigations (Table 1, Figure 1a,b).In Saxony, four locations were selected, including Michalken, Sparte am Moor, Waldbad Bernsdorf, and Senftenberger See.In Mecklenburg-West Pomerania, one location was selected, Hiddensee Island.In Brandenburg, nine locations were chosen, including Cottbus, Frauendorf, Geierwalder See, Deulowitzer See, Göhlensee, Heidesee, Pinnower See, Spremberg See, and Löhsten.In Löhsten, 14 sub-spots (Figure 2) were indicated by a hunter who was diagnosed with TBE in Carl-Thiem-Klinikum, Cottbus.During his visit to the forest, he left his car only at these sub-spots to check on hunting.In Lubush, two sites were selected, incuding Jezioro Głębokie and Jeziory Wysokie.In Lower Silesia, there were three locations chosen-Dziewiętlin, Kamienna, and Marianówka.All three sites in Lower Silesia were indicated as locations of repeated tick bites near the places of residence.The authors were not able to obtain any patient data from Western Pomerania (Poland); thus, no flagging site was selected there.At the chosen sites, ticks were collected from vegetation using a flagging method.Sites were visited from spring 2021 to summer 2022.Additionally, one partially engorged tick specimen was collected from a TBE patient at a hospital (Carl-Thiem-Klinikum Cottbus, Cottbus, Germany), and 41 non-engorged and partially engorged ticks were collected from a dog (Waldbad Bernsdorf, Germany).The collected ticks were identified life stage and species level [21,22] and stored at −80 • C until further tests.

Molecular Detection
Subsequently, due to the expected low prevalence of TBEV, ticks were tested in pools of a maximum of 10 specimens (per site, species, and life stage).Before RNA extraction, ticks were crushed with three rounds at a speed of 6.5 m/s for 30 s in the Fast Prep Savant FP120 tissue lyser (Bio101, Vista, CA, USA) in 1 mL minimum essential medium (MEM, Invitrogen, Karlsruhe, Germany) containing an antibiotic, antimycotic solution (ABAM, Invitrogen, Karlsruhe, Germany) [20].Then, the nucleic acid was extracted using the MagNA Pure LC Total Nucleic Acid Kit (Roche, Mannheim, Germany) in the MagNA Pure LC instrument (Roche, Mannheim, Germany) according to the manufacturer's instructions, with 200 µL of the tick homogenate supernatant.The extracted samples were tested for the presence of TBEV using a real-time RT-PCR (RT-qPCR) targeting the 3′noncoding region of the viral genome and 5 µL of the eluted RNA [23].For virus isolation, a 100 µL aliquot of the supernatants of each crushed RT-qPCR-positive tick pool was added to an 80% confluent cell culture of A549 cells (human lung carcinoma cells, German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany).The homogenates were kept at −80 °C until they were used undiluted and in a dilution of 1:5 and 1:10 for virus isolation.After 1 h of incubation at 37 °C, the supernatant was decanted, and the cells were washed 5 times with MEM containing ABAM.A total of 5 mL of MEM containing 5-fold concentrated ABAM and 3% fetal calf serum were added.Cells were incubated for up to 7 days at 37 °C and observed daily for the occurrence of cytopathogenic effect (cpe).In the case of more than 50% cpe, the supernatant was taken and tested using RT-qPCR for TBEV, as described [23].In case of no cpe, culture supernatant was taken after 7 days of incubation and also tested for the growth of TBEV using RT-qPCR.No subcultures were conducted.Based on previous experience, sequencing attempts directly from tick samples were performed only if the Ct value was <35.From the isolated TBEV strains, E genes were sequenced for confirmation, as described [20].

Molecular Detection
Subsequently, due to the expected low prevalence of TBEV, ticks were tested in pools of a maximum of 10 specimens (per site, species, and life stage).Before RNA extraction, ticks were crushed with three rounds at a speed of 6.5 m/s for 30 s in the Fast Prep Savant FP120 tissue lyser (Bio101, Vista, CA, USA) in 1 mL minimum essential medium (MEM, Invitrogen, Karlsruhe, Germany) containing an antibiotic, antimycotic solution (ABAM, Invitrogen, Karlsruhe, Germany) [20].Then, the nucleic acid was extracted using the MagNA Pure LC Total Nucleic Acid Kit (Roche, Mannheim, Germany) in the MagNA Pure LC instrument (Roche, Mannheim, Germany) according to the manufacturer's instructions, with 200 µL of the tick homogenate supernatant.The extracted samples were tested for the presence of TBEV using a real-time RT-PCR (RT-qPCR) targeting the 3 ′ -noncoding region of the viral genome and 5 µL of the eluted RNA [23].For virus isolation, a 100 µL aliquot of the supernatants of each crushed RT-qPCR-positive tick pool was added to an 80% confluent cell culture of A549 cells (human lung carcinoma cells, German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany).The homogenates were kept at −80 • C until they were used undiluted and in a dilution of 1:5 and 1:10 for virus isolation.After 1 h of incubation at 37 • C, the supernatant was decanted, and the cells were washed 5 times with MEM containing ABAM.A total of 5 mL of MEM containing 5-fold concentrated ABAM and 3% fetal calf serum were added.Cells were incubated for up to 7 days at 37 • C and observed daily for the occurrence of cytopathogenic effect (cpe).In the case of more than 50% cpe, the supernatant was taken and tested using RT-qPCR for TBEV, as described [23].In case of no cpe, culture supernatant was taken after 7 days of incubation and also tested for the growth of TBEV using RT-qPCR.No subcultures were conducted.Based on previous experience, sequencing attempts directly from tick samples were performed only if the Ct value was <35.From the isolated TBEV strains, E genes were sequenced for confirmation, as described [20].

Statistical and Phylogenetic Analyses
To estimate the prevalence of TBEV infection in ticks, the minimum infection rate (MIR) was used, i.e., the minimum infected proportion expressed as a percentage [24], as follows: where p = the number of positive pools, and N = the total number of ticks tested.It was assumed that if a pool tested positive, only one tick specimen in this pool was infected.Fisher's exact test was used to test for significant difference between two MIR estimates based on a two-tailed hypothesis [24].To test for significant differences in pathogen pool prevalence between sites or life stages, we used a chi-square test without continuity correction [25].The confidence intervals (95% CI), Fisher's test, and chi-square test were performed using Graph Pad Prism Software v. 4.0.(Graph Pad Software Inc., San Diego, CA, USA).The significance threshold was set at p = 0.05.
The sequence data were processed using the program Geneious 9.1.5(Biomatters, Auckland, New Zealand).A de novo assembly was conducted using the three chromatograms obtained from GATC (Eurofins Genomics, Ebersberg, Germany) for each positive sample.Nucleotides with an estimated error higher than 1% were trimmed.Subsequently, the sequences were cut to 1488 bp, the exact length of the envelope gene sequence.A ClustalW Alignment (GenomeNet, Kyoto University Bioinformatics Center, Kyoto, Japan) with several other E genes from selected isolates was performed, and a phylogenetic tree was generated using the PhyML 3.0 algorithm (Montpellier Bioinformatics, Montpellier, France) [26].
Ixodes ricinus ticks were found at almost all sites (Table 2, Figure 1), except for one in Brandenburg (Pinnower See, Germany), where only D. reticulatus ticks were collected.Dermacentor reticulatus was widely distributed in Brandenburg and Saxony (Germany), as well as in Lubush (Poland).It was absent, however, on an island in the northern part of Germany, Hiddensee Island (Mecklenburg, West Pomerania), and the southern part of Poland, a valley in the Sudetes mountain range, Marianówka, and Kamienna (Lower Silesia).Haemaphysalis concinna ticks were the most abundant in Saxony (all sites), and a few specimens were also found in Brandenburg and Lubush.Single individuals of I. frontalis were found at two sites in Saxony (Michalken and Senftenberger See), where all other tick species were collected, as well.
At Löhsten site, where MIR was the highest, a total of 669 ticks were collected from 14 sub-spots (Figure 2, Table 3).The dominant species there was D. reticulatus (96.6%; n = 646), followed by I. ricinus (2.4%; n = 16) and H. concinna (1%; n = 7).TBEV was detected in seven pools (MIR: 1.05%; 95% CI: 0.46-2.19)from two sub-spots (Figure 2, Table 4), including one pool of I. ricinus male, two pools of D. reticulatus females, and four pools of D. reticulatus males.At sub-spot no. 4, one pool was positive from 90 ticks collected (MIR: 1.1%; 95% CI: <0.01-6.6), and at sub-spot no. 10, six pools were positive from 160 ticks collected (MIR: 3.75%: 95% CI: 1.6-10.2).There were no significant differences in MIR between infected species D. reticulatus and I. ricinus (p = 0.158).At Löhsten site, where MIR was the highest, a total of 669 ticks were collected from 14 sub-spots (Figure 2, Table 3).The dominant species there was D. reticulatus (96.6%; n = 646), followed by I. ricinus (2.4%; n = 16) and H. concinna (1%; n = 7).TBEV was detected in seven pools (MIR: 1.05%; 95% CI: 0.46-2.19)from two sub-spots (Figure 2, Table 4), including one pool of I. ricinus male, two pools of D. reticulatus females, and four pools of D. reticulatus males.At sub-spot no. 4, one pool was positive from 90 ticks collected (MIR:   At Löhsten site, where MIR was the highest, a total of 669 ticks were collected from 14 sub-spots (Figure 2, Table 3).The dominant species there was D. reticulatus (96.6%; n = 646), followed by I. ricinus (2.4%; n = 16) and H. concinna (1%; n = 7).TBEV was detected in seven pools (MIR: 1.05%; 95% CI: 0.46-2.19)from two sub-spots (Figure 2, Table 4), including one pool of I. ricinus male, two pools of D. reticulatus females, and four pools of D. reticulatus males.At sub-spot no. 4, one pool was positive from 90 ticks collected (MIR: Unfortunately, virus cultivation was not successful for all positive pools, which may have been due to low virus load or the sample transport between different laboratories.From 19 positive pools, only twelve strains were successfully cultivated and sequenced, including ten from Germany-seven from Brandenburg, Löhsten (n = 5), Göhlensee (n = 2); three from Saxony, Sparte am Moor (n = 1) and Waldbad Bernsdorf (n = 2); and two from Poland, Lower Silesia and Marianówka.The phylogenetic analyses based on the E-gene revealed that all strains from Löhsten (Brandenburg) clustered the closest with German strains from Baden-Württemberg, Tübingen virus and Emmendingen (Figure 8).One sample from Göhlensee (Brandenburg) has the closest genetic relation to northeastern European strains from Finland and Estonia, and the second of the samples from the current study from Saxony.All isolates from Saxony (from both locations, Sparte am Moor and Walbad Bernsdorf) have the closest genetic relation to those from Baden-Württemberg, Wangen and Karsee.Samples from Poland (Lower Silesia, Marianówka) cluster the closest with German strains from North Rhine-Westphalia.

Discussion
The goals of the current study were the identification of new tick-borne encephalitis (TBE) microfoci, as well as the isolation and phylogenetic characterization of the tick-borne encephalitis virus (TBEV) strains circulating in nonendemic areas in Eastern Germany and Western Poland.Our strategy involved gathering information about potential locations from TBE patients who indicated the areas where they had most likely acquired tick bites.Subsequently, ticks were sampled in those locations for TBEV to increase the success of finding TBE microfoci.It must be mentioned that the research was conducted in 2021 and 2022, and due to the COVID-19 pandemic, some local health authorities were overwhelmed, and the notification of new TBE cases was delayed.However, despite these limitations, our approach seems to be an effective way of searching for new microfoci, as we were able to indicate five potential sites in Poland and fourteen in Germany.
We collected four tick species from the vegetation, including Ixodes ricinus, Dermacentor reticulatus, Haemaphysalis concinna (in Germany and Poland), and I. frontalis (only in Germany).These findings are in line with the distribution ranges of these species [21,[27][28][29].Interestingly, I. frontalis, an ectoparasite of birds that is usually collected from avian hosts, had been found on vegetation previously, also in Germany [30].
TBEV was detected in two tick species, I. ricinus and D. reticulatus, that are known vectors of TBEV in Europe [2,20,31,32].The overall minimal infection rate (MIR) of TBEV was low, 0.23%, with no significant differences between the two species.A previous study from a nonendemic area in Germany [20] had also shown no statistical difference in MIRs between I. ricinus and D. reticulatus, despite slightly higher rates (0.57% and 0.59%, respectively).It is suggested that D. reticulatus contributes, besides I. ricinus, to the virus expansion and helps to maintain circulation in new microfoci [31,33].
First-time virus detection in questing ticks was successful at six out of nineteen investigated locations.MIRs varied between microfoci from 0.13% to 1.05% and between I. ricinus life stages from 0.13% (nymphs) to 1.07% (females), which represents the general view of TBEV prevalence from previous European studies [24,[34][35][36][37][38][39][40].Additionally, we tested ticks removed from two hosts.The tick removed from the TBE patient was a partially engorged I. ricinus female, and from the dog (Waldbad Bernsdorf, Bernsdorf, Germany), 41 D. reticulatus ticks were removed (13 females and 28 males, all non-or partially engorged).However, despite a confirmed diagnosis, no TBEV could be amplified from the patient's tick or from the dog walking in the area where two pools were TBEV positive.TBE viremia is known to be short-lived and of low titer, which is why humans are considered dead-end hosts and attempts to detect TBEV in attached ticks usually fail [41].
We were able to identify six previously unknown microfoci in areas where human TBE cases have been reported, five in Germany (two in Brandenburg and three in Saxony) and one in Poland (Lower Silesia).Detailed coordinates of the microfoci are available in Table 1.Of these microfoci, three are located in districts that have recently been registered as TBE risk areas-Göhlensee (Oder-Spree County since 2022), Waldbad Bernsdorf, and Sparte am Moor (Bautzen County since 2018).The distance between these three microfoci is within 17 km, making it probable that the virus has spread directly from one microfocus to another through tick-infested or viremic hosts, such as mammals or birds [42].The microfocus Marianówka in Poland is located in Kłodzko County, which has the highest number of human cases (4-10 per year) in the Lower Silesia Voivodship (10-18 per year).The Löhsten microfocus with the highest MIR is situated less than 30 km away from a previously described TBEV microfocus (Battaune) [20].In Löhsten, TBEV was detected in two sub-sites (no. 4 and 10) that are 1.4 km apart.However, Löhsten and Battaune are in two different districts, and neither are considered TBE risk areas according to the federal Robert Koch Institute.Consequently, vaccination against TBE is not recommended in these areas, even though cases occur, and we have detected the virus presence there.
The TBEV sequences obtained from Löhsten (Brandenburg) show a close phylogenetic relation with strains from endemic areas in Baden-Württemberg, Tübingen virus and Emmendingen (located more than 500 km to the southwest), and the strains circulating in subsite no. 10 (not larger than 0.5 ha) most likely originated from two sources.There is no evidence that the isolates from Löhsten are related to those from the natural focus in Battaune, which are clustering with sequences from Poland and Bavaria (Neustadt an der Waldnaab, Germany) [20].TBEV in Göhlensee (Brandenburg)-the southeastern side of the lake-has also probably been introduced into the area at least twice independently.One sample has the closest genetic relationship with northeastern European strains from Finland and Estonia, which were most likely carried by birds, and the second one with the samples from microfoci that are located 70 km (Waldbald Bernsdorf) and 80 km (Sparte am Moor) to the south and could have been transported by ticks feeding on mammals, e.g., deer or wild boar, as well as birds [43,44].This suggests that two different strains are circulating in this location.Isolates from Saxony, microfoci Sparte am Moor and Walbad Bernsdorf, which are about 10 km apart, are closely related to each other, and the strains show the closest clustering with those from Baden-Württemberg, Wangen and Karsee (over 500 km southwest), implying a spread by migratory birds.Unfortunately, sequencing of the sample from the third microfocus in Saxony, Senftenberger See, was not successful.The TBEV sequences from Marianówka (Poland) show that the virus circulating in this microfocus is phylogenetically closely related to German strains from North Rhine-Westphalia.These strains are more than 600 km away in a westerly direction.
As for the rest of the sites where no TBEV-positive ticks were found, it is likely that the patients did not correctly recall where they had acquired tick bites or that we missed the right location when flagging.It is also very common for patients suffering from TBDs not to remember being bitten by a tick [45,46].

Conclusions
In summary, our results show that the patient-derived approach is successful in the search for new TBEV microfoci.The TBEV prevalence (MIR) within the study area was rather low.The detection of previously unknown TBEV microfoci highlights the risk of TBE even in nonendemic areas.Increased public awareness regarding vaccination and surveillance efforts are therefore required.The phylogenetic relationships presented for the newly isolated TBEV strains from Eastern Germany and Southwestern Poland indicate that it is not a simple geographical spread that links areas with TBEV outbreaks.In particular, the significance of the noncontinuous distribution of TBEV patterns over long distances is surprising.Genetic data suggested, among other things, an association with bird migration.

Figure 1 .
Figure 1.Tick collecting sites in Germany and Poland indicated by tick-borne encephalitis (TBE) patients (© StepMap, 123 map, Data: OpenStreetMap, License ODbL 1.0); (a) German states and Polish voivodships where the study was conducted (orange); (b) collection sites selected for the study (red pins).

Figure 1 .
Figure 1.Tick collecting sites in Germany and Poland indicated by tick-borne encephalitis (TBE) patients (© StepMap, 123 map, Data: OpenStreetMap, License ODbL 1.0); (a) German states and Polish voivodships where the study was conducted (orange); (b) collection sites selected for the study (red pins).

Figure 2 .
Figure 2. Site Löhsten (Germany, Brandenburg).Ticks were collected at 14 sub-spots indicated by a hunter.Tick-borne encephalitis virus was found at two sub-spots (red circle)-one pool at sub-spot no. 4 and six pools at sub-spot no. 10 (Google Maps with own modifications).

Figure 2 .
Figure 2. Site Löhsten (Germany, Brandenburg).Ticks were collected at 14 sub-spots indicated by a hunter.Tick-borne encephalitis virus was found at two sub-spots (red circle)-one pool at sub-spot no. 4 and six pools at sub-spot no. 10 (Google Maps with own modifications).

Figure 3 .
Figure 3. Site Göhlensee (Germany, Brandenburg).The flagging area is marked in transparent red, where six positive pools were found (Google Maps with own modifications).Figure 3. Site Göhlensee (Germany, Brandenburg).The flagging area is marked in transparent red, where six positive pools were found (Google Maps with own modifications).

Figure 3 .
Figure 3. Site Göhlensee (Germany, Brandenburg).The flagging area is marked in transparent red, where six positive pools were found (Google Maps with own modifications).Figure 3. Site Göhlensee (Germany, Brandenburg).The flagging area is marked in transparent red, where six positive pools were found (Google Maps with own modifications).

Figure 4 .
Figure 4. Site Senftenberger See (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 5 .
Figure 5. Site Waldbad Bernsdorf (Germany, Saxony).The flagging area is marked in transparent red.where two positive pools were found (Google Maps with own modifications).

Figure 4 .
Figure 4. Site Senftenberger See (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 4 .
Figure 4. Site Senftenberger See (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 5 .
Figure 5. Site Waldbad Bernsdorf (Germany, Saxony).The flagging area is marked in transparent red.where two positive pools were found (Google Maps with own modifications).Figure 5. Site Waldbad Bernsdorf (Germany, Saxony).The flagging area is marked in transparent red.where two positive pools were found (Google Maps with own modifications).

Figure 5 .
Figure 5. Site Waldbad Bernsdorf (Germany, Saxony).The flagging area is marked in transparent red.where two positive pools were found (Google Maps with own modifications).Figure 5. Site Waldbad Bernsdorf (Germany, Saxony).The flagging area is marked in transparent red.where two positive pools were found (Google Maps with own modifications).

Figure 6 .
Figure 6.Site Marianówka (Poland, Lower Silesia).The flagging area is marked in transparent red, where two positive pools were found (Google Maps with own modifications).

Figure 7 .
Figure 7. Site Sparte am Moor (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 6 .
Figure 6.Site Marianówka (Poland, Lower Silesia).The flagging area is marked in transparent red, where two positive pools were found (Google Maps with own modifications).

Figure 6 .
Figure 6.Site Marianówka (Poland, Lower Silesia).The flagging area is marked in transparent red, where two positive pools were found (Google Maps with own modifications).

Figure 7 .
Figure 7. Site Sparte am Moor (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 7 .
Figure 7. Site Sparte am Moor (Germany, Saxony).The flagging area is marked in transparent red, where one positive pool was found (Google Maps with own modifications).

Figure 8 .
Figure 8. Phylogenetic tree for tick-borne encephalitis virus (TBEV) based on E-gene sequences.Samples from this study are marked in bold.Figure 8. Phylogenetic tree for tick-borne encephalitis virus (TBEV) based on E-gene sequences.Samples from this study are marked in bold.

Figure 8 .
Figure 8. Phylogenetic tree for tick-borne encephalitis virus (TBEV) based on E-gene sequences.Samples from this study are marked in bold.Figure 8. Phylogenetic tree for tick-borne encephalitis virus (TBEV) based on E-gene sequences.Samples from this study are marked in bold.

Table 1 .
Location and description of 19 potential tick-borne encephalitis (TBE) microfoci selected for investigations.

Table 2 .
The number of collected ticks at 19 flagging sites and from 2 hosts (the tick-borne encephalitis patient at a hospital and the dog from one of the flagging sites).
* Ticks collected from hosts.

Table 3 .
Tick-borne encephalitis virus-positive pools and minimum infection rates (MIRs) detected in ticks.

Table 3 .
Tick-borne encephalitis virus-positive pools and minimum infection rates (MIRs) detected in ticks.