The proposed thesis contributes to the basic knowledge in tick (Ixodes ricinus) and tick-borne pathogens (Borrelia burgdorferi sensu lato, tick-borne encephalitis virus) ecology in particular studying the spatial distribution, host associations and its causes and consequences in Central European habitats.

The first aim of this study was to detect antiviral activities of substances isolated from natural products against tick-borne encephalitis virus in in vitro model. Resveratrol isolated from plant material and adamantane derivatives were studied in this regard. The maximum tolerated concentrations of the investigated substances were determined for the glioblastoma cell line used in the experiments using flow cytometry and subsequently. Next, the number of viral particles produced by infected cells after incubation with the studied substances was determined using plaque titration. Possible antibacterial effects of the studied materials against standard strains of bacteria Staphyloccocus aureus, Staphyloccocus epidermidis, Escherichia coli and selected strains of Borrelia burgdorferi spirochetes were examined.

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study’s overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.

Fästingburet encefalitvirus (TBEV) är ett RNA-virus som tillhör genuset flavivirus. Vid en TBEV-infektion är feber, trötthet, allmänpåverkan samt huvudvärk och muskelvärk vanligt förekommande symtom. Viruset överförs via saliven från fästingar under de första minuterna efter fästingbett. TBEV-IgM och ibland även TBEV-IgG återfinns i serum då symtom i centrala nervsystemet (CNS) yttrar sig i den andra fasen av sjukdomsförloppet. De senaste åren har prevalensen av fästingburen encefalit (TBE) ökat. Sedan 2017 har över 300 fall av TBE rapporterats årligen i Sverige. Laterala flödesanalyser (lateral flow assays, LFA) är billiga, enkla, snabba och baseras på portabla instrument som används bland annat inom biomedicinsk vetenskap. ReaScan® TBE IgM från det finska företaget Reagena är ett snabbtest, baserat på LFA-tekniken, för detektion av TBE-specifika IgM-antikroppar i humant serum och likvor. Syftet med studien var att undersöka om ReaScan® TBE IgM kan användas för att diagnostisera TBE på laboratoriet för Klinisk Mikrobiologi på länssjukhuset i Kalmar. Metodens prestanda undersöktes genom att analysera totalt 23 serumprover, 13 prover från TBE-patienter och 10 prover från icke-TBE-patienter. Sensitiviteten uppskattades genom att analysera 13 serumprover där förekomst av TBE-antikroppar sedan tidigare konfirmerats. Specificiteten uppskattades genom att analysera 10 serumprover från patienter utan känd TBEV-infektion. Den diagnostiska sensitiviteten respektive specificiteten beräknades till 100 %. På grund av den begränsade storleken på undersökningsmaterialet är dock den beräknade sensitiviteten och specificiteten ej helt tillförlitlig. Metodens prestanda ansågs vara tillräckligt god för att den skall kunna användas som en screening-metod för TBEV-IgM-antikroppar på laboratoriet för Klinisk Mikrobiologi på länssjukhuset i Kalmar. / Tick-borne encephalitis virus (TBEV) is an RNA virus that belongs to the genus flavivirus. Symptoms that commonly present during a TBEV infection include headaches, muscle pains, fever and malaise. The virus is transmitted with the saliva from ticks during the first minutes of their blood meal. TBEV-IgM and sometimes TBEV-IgG antibodies can be detected in the patient’s serum when central nervous system (CNS) symptoms present in the second phase of the disease. Over the last couple of years, the prevalence of tick-borne encephalitis (TBE) has increased. Since 2017 over 300 cases of TBE are reported every year in Sweden. Lateral flow assays (LFA) is the technology behind inexpensive, simple, quick and portable instruments that are used within the biomedical science field among others. ReaScan® TBE IgM developed by the Finnish company Reagena is a rapid test, based on the LFA technique, used for the detection of TBEV specific IgM antibodies in human serum and cerebrospinal fluid. The trial aimed to evaluate whether ReaScan® TBE IgM could be used to diagnose TBE at the laboratory of Clinical microbiology at the County hospital in Kalmar. The performance of the test was determined by analysing a total of 23 serum samples, 13 of which consisted of samples from patients with a previously confirmed TBE diagnosis and 10 samples from patients with no known TBEV infection. The diagnostic sensitivity and specificity were both determined to be 100 %. Due to the limited sample size, the calculated sensitivity and specificity are not particularly reliable.  The performance of the test was satisfactory and it could be used as a screening method for the detection of TBEV IgM antibodies at the department of Clinical microbiology at Kalmar County Hospital.

Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.

Video S1.mov: Co-localization of TBEV and EEA1Video S2.mov: Co-localization of TBEV and SNX5Video S3.mov: Tight junction changes induced by TBEV infection / Das Frühsommer-Meningoezephalitis-Virus (FSMEV) ist eines der wichtigsten vektorübertragenen Viren in Europa und Asien. Die häufigste Übertragung erfolgt durch den Stich einer infizierten Zecke, gelegentlich werden FSME Infektionen auch durch den Genuss von Rohmilchprodukten infizierter Tiere verursacht. Die Pathogenese von Caco-2 Monolayer Epithelzellen zeigten nach Infektion mit FSMEV morphologische Änderungen mit signifikanter Vakuolisierung. Ultrastrukturanalysen zeigten eine Ausdehnung des rauen ER und das Auftreten FSMEV haltiger Kavernen. Monolayer von Caco-2 Zellen bildeten eine Barriere mit stabilem transepithelialem elektrischem Widerstand (TEER). Auch traten Viren im basolateralen Medium auf, die über einen Tanscystose pathway (PW) aufgenommen wurden. Der Zelleintritt von FSMEV konnte durch verschiedene Inhibitoren wirksam blockiert werden, was darauf hinweist, dass Aktinfilamente und Mikrotubuli wichtig für die PI3K-abhängige Endozytose sind. Die experimentelle Flüssigkeitsaufnahme zeigte erhöhte intrazelluläre Ansammlungen von FITC-Dextran haltigen Vesikeln und die Co-Lokalisation von FSME-Viren mit frühem Endosom Antigen-1 und mit sorting nexin-5. Was auf die Makropinozytose als Transportmechanismus hinweist. Während der Infektion wurden weitere Hinweise für die Virustranslokation über den parazellulären Weg gefunden. Das konnte den FSMEV Pathomechanismus in humanen Intestinalepithelzellen über Nahrungsmittel näher aufklären. Die Untersuchung der zwei UPR „signaling PWs“ während der FSMEV Infektion in VeroE6 Zellen zeigte, dass die Menge von „heat shock protein“ 72 im Verlauf der FSMEV Infektion ansteigt, und eine FSMEV Infektion den „IRE1- und den ATF6 PW“ aktiviert. Auch die Hemmung des „IRE1 PW“ wirkt auf die FSMEV Infektion, was darauf hinweist, dass eine FSMEV Infektion die beiden „UPR signaling PWs“ aktiviert. Diese Hemmung der FSMEV Replikation durch UPR Inhibitoren könnte ein neuer Ansatz für spezifische Therapien gegen FSME sein. / Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. The transmission mainly occurs by the bite of an infected tick. Consuming of rough milk products from infected livestock animals also occasionally cause TBE cases. Human intestinal Caco-2 cells were used to investigate the pathogenesis caused by TBEV. During TBEV infection Caco-2 monolayers showed morphological changes with significant vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers showed an intact epithelial barrier with stable transepithelial electrical resistance (TER). Concomitantly, viruses were detected in the basolateral medium, taken up via a transcytosis pathway. TBEV cell entry was efficiently blocked with different inhibitors, suggesting that actin filaments and microtubules are important for PI3K-dependent endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles and co-localization of TBEV with early endosome antigen-1 and with sorting nexin-5 could confirm macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Thus, TBEV pathomechanisms in human intestinal epithelial cells and its transmission via the alimentary route were enlightened. In addition, I investigated the effects of the two unfolded protein response (UPR) signaling pathways upon TBEV infection in Vero E6 cells. I showed that the amount of heat shock protein 72 increased in the course of TBEV infection. I then confirmed that TBEV infection activates the IRE1 pathway and ATF6 pathway. These findings provide the first evidence that TBEV infection activates the two UPR signaling pathways. Moreover, inhibition of UPR may provide a novel therapeutic strategy against TBE.

No description available.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.