Tick-borne Encephalitis Research Articles

Unraveling the role of human microglia in tick-borne encephalitis virus infection: insights into neuroinflammation and viral pathogenesis

Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly targeted, specific involvement of microglia in pathogenesis of TBE is not yet fully understood. In this study, the susceptibility of human microglia to TBEV is investigated, focusing on productive infection and different immune responses of different viral strains. We investigated primary human microglia and two immortalized microglial cell lines exposed to three TBEV strains (Hypr, Neudörfl and 280), each differing in virulence. Our results show that all microglia cultures tested support long-term productive infections, regardless of the viral strain. In particular, immune response varied significantly with the viral strain, as shown by the differential secretion of cytokines and chemokines such as IP-10, MCP-1, IL-8 and IL-6, quantified using a Luminex 48-plex assay. The most virulent strain triggered the highest cytokine induction. Electron tomography revealed substantial ultrastructural changes in the infected microglia, despite the absence of cytopathic effects. These findings underscore the susceptibility of human microglia to TBEV and reveal strain-dependent variations in viral replication and immune responses, highlighting the complex role of microglia in TBEV-induced neuropathology and contribute to a deeper understanding of TBE pathogenesis and neuroinflammation.

Clinical and laboratory prognostic factors in patients with mixed infection of erythema form of ixodic tick-borne borreliosis and tick-borne encephalitis

Background. Early diagnosis of the mixed infection of ixodic tick-borne borreliosis and tick-borne encephalitis can be difficult without laboratory confirmation of the diagnosis.The aim. To assess clinical and laboratory predictors in patients for building the models of early differential diagnosis between mixed infection of erythema form of ixodic tick-borne borreliosis and febrile form of tick-borne encephalitis and monoinfection of erythema form of ixodic tick-borne borreliosis.Materials and methods. The clinical study included 26 patients with mixed infection of the erythema form of ixodic tick-borne borreliosis with tick-borne encephalitis (mean age – 46.7 years) and 30 patients with monoinfection of the erythema form of ixodic tick-borne borreliosis (mean age – 48.5 years). 59 clinical, epidemiological and laboratory parameters, including 6 leukocyte blood indices, standard and extended hemogram profiles were studied in these patients the first week of the disease. For statistical analysis, we used Pearson and Mann – Whitney goodness-of-fit tests, ROC analysis with calculation of the area under curve (AUC) and logistic regression. Results. Patients with mixed infection, compared to the patients with monoinfection, had increased odds of having febrile fever, weakness, headache and orbital pain; meningism symptoms occurred only in patients with mixed infection. Early predictors of a preliminary diagnosis of mixed infection were elevated values of fever (AUC = 0.87), intoxication syndrome score (AUC = 0.71), leukocyte count (AUC = 0.64) and neutrophils (AUC = 0.71) in the hemogram, levels of leukocyte intoxication index according to V.K. Ostrovsky (AUC = 0.67) and neutrophil-leukocyte index (AUC = 0.65), as well as a decrease in the number of platelets (AUC = 0.77), eosinophils (AUC = 0.72) and basophils (AUC = 0.79) in peripheral blood. Two prognostic models which included clinical and laboratory parameters demonstrated excellent prediction of mixed infection (AUC = 0.90 and AUC = 0.91).Conclusion. Early clinical and laboratory predictors have been established and models have been developed that make it possible, with excellent prognosis quality, to calculate in patients the probability of a preliminary diagnosis of mixed infection of the erythema form of ixodic tick-borne borreliosis with tick-borne encephalitis in comparison with borreliosis monoinfection.

TBE In Italy

Italy is considered a low-incidence country for tick-borne encephalitis (TBE) in Europe.1 Areas at higher risk for TBE within Italy are geographically clustered in the forested and mountainous regions and provinces of the northeastern part of the country, as suggested by TBE case series published over the last decade. 2–4 A national enhanced surveillance system for TBE has been established since 2017.5 Before this, information on the occurrence of TBE cases at the national level in Italy was lacking. Both incidence rates and the geographical distribution of the disease were mostly inferred from endemic areas where surveillance was already in place, and from ad hoc studies and international literature. TBE has been recorded in Italy since 1967, with foci of infections in the northeast (Trento, Belluno and Gorizia) and central (Florence and Latina) provinces.6–9 TBE presence in central Italy has not been confirmed by further studies on ticks and serosurveys conducted afterwards,10,11 nor by human cases, posing concerns about possible misdiagnosis.

Clinical characteristics and factors affecting disease severity in hospitalized tick-borne encephalitis patients in Norway from 2018 to 2022

PurposeTo describe the clinical characteristics and factors associated with disease severity in a Norwegian cohort of hospitalized patients with tick-borne encephalitis (TBE).MethodsThis observational multicenter study included hospitalized patients with TBE in the endemic area in the southeastern region of Norway from 2018 to 2022. Clinical signs and findings from laboratory tests, EEG, CT and MRI scans were recorded. Patient characteristics were compared among those with mild, moderate, and severe TBE, and factors associated with disease severity were identified.ResultsNearly all eligible patients were included in the final cohort (153/189 participants, 81%). The median age was 56 years, 63% were men, and 7% were vaccinated against TBE; no participants were fully vaccinated. TBE presented as mild (meningeal) disease in 31% of patients and as moderate or severe (encephalitic) disease in 54% and 14% of patients, respectively. We found that 46% of the patients had a monophasic course, 64% had hyponatremia, and 7% presented with central nervous system (CNS) symptoms without pleocytosis in cerebrospinal fluid (CSF). Dysesthesia, a symptom previously not described, was reported in 10% of the patients. Most objective findings were related to the CNS. Preexisting comorbidities, CRP and CSF protein levels were predictors of more severe disease.ConclusionThis novel presentation of a large Norwegian cohort supports TBE as a serious disease in the southeastern region of Norway. The majority of hospitalized patients presented with encephalitis, and fewer presented with meningitis. Comorbidities, CRP and CSF protein levels were associated with more severe disease.Trial registrationProsjekt #2,296,959 – The Norwegian Tick-borne Encephalitis Study – NOTES. Acute phase characteristics and long-term outcomes. – Cristin.

TBE In Poland

The history of tick-borne encephalitis (TBE) in Poland started in 1948, when clinical symptoms of TBE were described by Demiaszkiewicz.7 Disease reporting has been mandatory since 1970. In the years between 1970-1992, a total of 576 TBE cases were reported; the annual number varied from 4 (1991) to 60 (1970), and the incidence in that period ranged from 0.01/100,000 population to 0.2/100,000 inhabitants, respectively. In 1993, however, the number of reported TBE cases increased rapidly, probably because of the first introduction of commercial tests serologically to confirm the diagnosis of TBE by ELISA, which rapidly replaced the older HI assay (Fig.1).2,3,15 As in other European countries, TBE cases occur mainly in men aged 30-60 y. (Fig.2).

TBE in the Netherlands

Until 2015, tick-borne encephalitis virus (TBEV) was presumed not to be endemic in the Netherlands.1,2 Consequently, the number of diagnostic requests for detection of tick-borne encephalitis (TBE) infection has been low. Between 2006 and 2015, the laboratory of the Netherlands Centre for Infectious Disease Control (CIb), 1 of the 2 laboratories that performed TBEV diagnostics in the Netherlands at the time, received an average of 20 (range 12–27) requests for TBEV diagnostics per year. In the same period, TBE was diagnosed in 7 Dutch patients. All cases were considered to be travel-related. Indeed, 6 out of 7 patients reported that they had recently travelled to TBEV-endemic countries such as Austria (4), Germany (1), and Sweden (1).

TBE In Estonia

The first cases of tick-borne encephalitis (TBE) in Estonia were identified in 1949. Today, Estonia is a TBE- endemic country. A TBE-endemic area in Estonia is defined as an area with circulation of the TBEV between ticks and vertebrate hosts as determined by detection of the TBEV or the demonstration of autochthonous infections in humans or animals within the last 20 years.

TBE in China

Tick-borne encephalitis (TBE) is an endemic disease in some regions of northern China. The first TBE patients were reported in 1943 and TBE virus (TBEV) was isolated from brain tissues of two patients in 1944 by Japanese military scientists1 as well as from patients and ticks (I. persulcatus and Haemaphysalis concinna) in 1952 by Chinese researchers.2

TBE In Denmark

Since the 1950s tick-borne encephalitis (TBE) has been endemic in Denmark but only on the island of Bornholm. Bornholm is situated east of mainland Denmark, south of Sweden (Figure 3) and has a different fauna and flora from the rest of Denmark. Bornholm has about 45,000 inhabitants, but about 500,000 tourists visiting every year.

TBE In Lithuania

The first case of tick-borne encephalitis (TBE) in Lithuania, diagnosed by clinical and epidemiologic criteria only, was reported in 1953. A forest worker became ill with the disease in April after a tick bite, had a typical clinical presentation with shoulder girdle muscle paralysis and bulbar syndrome, and died after 12 days from the start of clinical symptoms. Autopsy data were compatible with viral encephalitis.1 Serological diagnosis of TBE in Lithuania was started in 1970.2

TBE in Norway

In Norway, tick-borne encephalitis (TBE) has been a mandatory notifiable disease since 1975 (Norwegian Surveillance system for communicable diseases, MSIS).1 According to ECDCs classification, coastal areas in southern Norway (counties of Agder, Vestfold and Telemark) are endemic for TBE. Further, the counties of Østfold, Akershus and Buskerud, and western and northern Norway to Brønnøy municipality are imperiled.2-9

TBE In Bulgaria

First cases of probable tick-borne encephalitis (TBE) were reported in 1961 by Andonov et al. in eastern regions of Bulgaria.1 Possible TBE cases with the typical two-wave fever, originating from consumption of raw goat milk, were described back in 1953 by Vaptzarov et al. in southern Bulgaria.2 Investigations in the 1960s were able to isolate 3 tick-borne encephalitis virus (TBEV) strains from Haemaphysalis punctata and 1 from Dermacentor marginatus ticks from goats and sheep in the district of Plovdiv.3 The antigenic properties of these 4 virus strains were identical to the highly virulent strain “Hypr” of the European subtype of TBEV (TBEV-EU).3

TBE In Czech Republic

The TBE virus (TBEV) was first isolated in the Czech Republic by a Czech scientist in 1948-1949 from both a patient and also from Ixodes ricinus ticks.1 However, even before 1948, etiologically unclear summer cases of viral meningoencephalitis had been reported, and likely, at least in part, they are attributable to the TBEV. These cases were reported mostly from patients in the districts of Beroun (Central Bohemia), Hradec Králové (East Bohemia), Vyškov (South Moravia), and occasionally from the neighborhood of Prague. The official reports of these probable cases of “tick-borne encephalitis” were registered in the database of the National Institute of Public Health in Prague since 1945.

TBE In Moldova

Tick-borne encephalitis is monitored in the Republic of Moldova with an emphasis on surveillance of ticks with no attention to human cases. The competent national institution responsible for TBE monitoring is the National Agency for Public Health. Official data on vector testing have been recorded since 2011. Ever since, studies on the circulation of the TBEV are conducted annually in spring, summer and autumn by collecting ticks from several regions of the country: Floreşti, Soroca, Bender, Tiraspol, Orhei, Drochia, Hînceşti, Ialoveni, Străşeni, Vadul lui Vodă, Chişinău, Taraclia, and Comrat territorial administrative units. TBEV was detected in most of the regions mentioned above, with highest isolation rates in Chişinău municipality (including Vadul lui Vodă), Străşeni, Comrat, Bender, and Tiraspol (Figure 1).

TBE In France

The first human case of tick-borne encephalitis virus (TBEV) infection in France was reported in 1968 in Alsace, an eastern region next to the German border: a gamekeeper working in a forest near Strasbourg.1 Between 1970 and 1974, an extensive research survey confirmed the presence of TBEV in ticks and rodents in this French region. Eight percent of adult tick batches collected were infected (Ixodes ricinus) by the TBEV. Tick collection occurred in a forest near Strasbourg, the main city in the region. Nymphs were more rarely infected (1.6% of the collected lots).1 These data were confirmed in 2011 in Alsace in Guebwiller’s Valley, a middle altitude forest, with identification of western (European) subtype TBEV (TBEV-EU). The infection rate still remains low: TBEV was detected only in the I. ricinus nymphs (2.48%) that were collected during May; however, not in those collected during the other spring or summer months. In a more recent study, Bestehorn et al., collected ticks (953 male, 856 female adult ticks and 2,255 nymphs) in endemic foci in the upper Rhine region in France and Germany between 2016, 2017 and 2018 by flagging2. The minimal infection rate (MIR) of the collected ticks in the Foret de la Robertsau (France) was estimated to 0,11% (1 nymph/944 ticks). The isolated and sequenced TBEV strain from Foret de la Robertsau (F) is related to circulating TBEV isolates from eastern Bavaria and the Czech Republic. In the French department Alsace, there are today at least two independent TBEV strains circulating: the historical Alsace strain isolated in 1971 and the newly identified strain from Foret de la Robertsau. Other wooded regions (Ardennes) were explored for TBEV in ticks, but without evidence of virus infection.3

Exposure of cattle to tick-borne encephalitis virus in the historical endemic zone in north-eastern France

BackgroundTick-borne encephalitis (TBE) is a severe human neuroinfection caused by TBE virus (TBEV). TBEV is transmitted by tick bites and by the consumption of unpasteurized dairy products from infected asymptomatic ruminants. In France, several food-borne transmission events have been reported since 2020, raising the question of the level of exposure of domestic ungulates to TBEV. In this study, our objectives were (i) to estimate TBEV seroprevalence and quantify antibodies titres in cattle in the historical endemic area of TBEV in France using the micro virus neutralisation test (MNT) and (ii) to compare the performance of two veterinary cELISA kits with MNT for detecting anti-TBEV antibodies in cattle in various epidemiological contexts. A total of 344 cattle sera from four grid cells of 100 km² in Alsace-Lorraine (endemic region) and 84 from western France, assumed to be TBEV-free, were investigated.ResultsIn Alsace-Lorraine, cattle were exposed to the virus with an overall estimated seroprevalence of 57.6% (95% CI: 52.1–62.8%, n = 344), varying locally from 29.9% (95% CI: 21.0–40.0%) to 92.1% (95% CI: 84.5–96.8%). Seroprevalence did not increase with age, with one- to three-year-old cattle being as highly exposed as older ones, suggesting a short-life duration of antibodies. The proportion of sera with MNT titres lower than 1:40 per grid cell decreased with increased seroprevalence. Both cELISA kits showed high specificity (> 90%) and low sensitivity (less than 78.1%) compared with MNT. Sensitivity was lower for sera with neutralising antibodies titres below 1:40, suggesting that sensitivity of these tests varied with local virus circulation intensity.ConclusionsOur results highlight that cattle were highly exposed to TBEV. Screening strategy and serological tests should be carefully chosen according to the purpose of the serological study and with regard to the limitations of each method.

Potential drivers of vector-borne pathogens in urban environments: European hedgehogs (Erinaceus europaeus) in the spotlight

Vector-borne diseases (VBDs) are considered as (re-)emerging, but information on the transmission cycles and wildlife reservoirs is often incomplete, particularly with regard to urban areas. The present study investigated blood samples from European hedgehogs (Erinaceus europaeus) presented at wildlife rehabilitation centres in the region of Hanover. Past exposure to B. burgdorferi sensu lato (s.l.) and tick-borne encephalitis virus (TBEV) was assessed by serological detection of antibodies, while current infections with Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Neoehrlichia mikurensis, Bartonella spp., Babesia spp. and Spiroplasma ixodetis were investigated by (q)PCR. Of 539 hedgehogs tested for anti-Borrelia antibodies, 84.8% (457/539) were seropositive, with a higher seropositivity rate in adult than subadult animals, while anti-TBEV antibodies were detected in one animal only (0.2%; 1/526). By qPCR, 31.2% (168/539) of hedgehog blood samples were positive for Borrelia spp., 49.7% (261/525) for A. phagocytophilum, 13.0% (68/525) for Bartonella spp., 8.2% for S. ixodetis (43/525), 8.0% (42/525) for Rickettsia spp. and 1.3% (7/525) for Babesia spp., while N. mikurensis was not detected. While further differentiation of Borrelia spp. infections was not successful, 63.2% of the A. phagocytophilum infections were assigned to the zoonotic ecotype I and among Rickettsia spp. infections, 50.0% to R. helvetica by ecotype- or species-specific qPCR, respectively. Sequencing revealed the presence of a Rickettsia sp. closely related to Rickettsia felis in addition to a Bartonella sp. previously described from hedgehogs, as well as Babesia microti and Babesia venatorum. These findings show that hedgehogs from rehabilitation centres are valuable sources to identify One Health pathogens in urban areas. The hedgehogs are not only exposed to pathogens from fleas and ticks in urban areas, but they also act as potent amplifiers for these vectors and their pathogens, relevant for citizens and their pets.

In vivo rescue of arboviruses directly from subgenomic DNA fragments

ABSTRACT Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the “Infectious Subgenomic Amplicons” (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo. Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo. It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Different receptor models show differences in ligand binding strength and location: a computational drug screening for the tick-borne encephalitis virus.

The tick-borne encephalitis virus (TBE) is a neurotrophic disease that has spread more rapidly throughout Europe and Asia in the past few years. At the same time, no cure or specific therapy is known to battle the illness apart from vaccination. To find a pharmacologically relevant drug, a computer-aided drug screening was initiated. Such a procedure probes a possible binding of a drug to the RNA Polymerase of TBE. The crystal structure of the receptor, however, includes missing and partially modeled regions, which rendered the structure incomplete and of questionable use for a thorough drug screening procedure. The quality of the receptor model was addressed by studying three putative structures created. We show that the choice of receptor models greatly influences the binding affinity of potential drug molecules and that the binding location could also be significantly impacted. We demonstrate that some drug candidates are unsuitable for one model but show decent results for another. Without any prejudice on the three employed receptor models, the study reveals the imperative need to investigate the receptor structure before drug binding is probed whether experimentally or computationally.