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.

Les Flavivirus neuropathogènes, tels que le virus de l’encéphalite japonaise (JEV), le virus West Nile (WNV), le virus de la fièvre jaune (YFV) et le virus Zika (ZIKV) causent des maladies neurologiques. Ces maladies sont dues à une infection des cellules du système nerveux central (CNS) par ces virus. Le CNS est un organe privilégié, isolé des agents pathogènes par une barrière entre le sang et le cerveau, appelée barrière hémato-encéphalique (BBB). Les Flavivirus neuropathogènes capables de traverser cette BBB afin d’atteindre leurs cellules cibles, localisées dans le CNS, sont neuroinvasifs. Le but de cette étude est de comprendre les mécanismes cellulaires permettant aux Flavivirus de traverser la BBB et les effets de l’infection par les virus ZIKV et WNV des cellules du CNS sur le développement de celles-ci.Le YFV est un virus hépatotrope, infectant majoritairement le foie et les reins. Deux vaccins vivants atténués dirigés contre le YFV, le vaccin FNV (pour French Neurotropic Virus) et le vaccin 17D, ont été obtenus empiriquement par passages successifs de souches virulentes de YFV sur cerveaux de souriceaux. Ces vaccins ne causent plus de maladies touchant les reins et le foie, mais peuvent parfois causer des encéphalites post-vaccinales. Ces cas d’encéphalites démontrent que ces souches vaccinales sont devenues neurovirulentes mais aussi neuroinvasives car les virus ont pu franchir la BBB. A cause d’une incidence trop élevée d’encéphalites post-vaccinales par rapport au vaccin 17D, le vaccin FNV a été retiré du marché dans les années 1980.Le JEV est un virus neurotrope, causant des encéphalites graves en Asie du Sud-Est. A ce jour, il existe un vaccin vivant atténué, le JEV SA14-14-2, obtenu empiriquement par passages successifs d’une souche virulente sur cellules de hamster. Ce vaccin est moins neurovirulent et moins neuroinvasif que les souches virulentes de JEV en modèle de souris, et protège contre des infections humaines par le JEV. Cependant, des cas d’encéphalites ont été rapportés après injection de ce vaccin. Il apparait donc que, dans certains cas, la souche vaccinale JEV SA14-14-2 est capable de traverser la BBB et d’infecter les cellules neuronales. Les dernières épidémies à virus ZIKV en Polynésie Française et en Amérique du Sud ont induit une augmentation de cas de malformations congénitales dans les zones touchées. Cela a soulevé de nouvelles questions quant à la capacité d’un Flavivirus à provoquer des malformations congénitales du CNS. Dans cette étude, nous avons identifié les mécanismes cellulaires permettant aux Flavivirus de traverser la BBB et les effets de l’infection par les virus ZIKV et WNV des cellules du CNS sur le développement de celles-ci.Nous avons utilisé deux systèmes in vitro permettant d’étudier le développement du CNS et la neuroinvasion des Flavivirus. Un premier système consiste en l’infection de coupes de cerveaux d’embryon de souris. En utilisant ce système, nous avons montré que le ZIKV a un tropisme préférentiel pour les cellules progénitrices de neurones, alors que le WNV a un tropisme préférentiel pour les neurones. Nous avons également montré que l’infection des progéniteurs neuronaux par le ZIKV induit un arrêt de la mitose cellulaire, alors que l’infection par le WNV n’a aucun effet sur la mitose. L’étude sur l’effet apoptotique de l’infection par les deux virus WNV et ZIKV n’a montré aucune différence entre les deux virus à des temps précoces d’infection.Un deuxième système a été mis au point pour l’étude de la neuroinvasion par les Flavivirus neuropathogènes. Ce système est composé de cellules endothéliales hCMEC/D3 pouvant former des jonctions serrées. Ces cellules ont été cultivées sur filtres d’insert de puits de culture cellulaire Transwell, placés au-dessus de cellules neuronales humaines. A l’aide de ce système, nous avons comparé la capacité à traverser la BBB de plusieurs Flavivirus. / Neuropathogenic Flaviviruses, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV) and Zika virus (ZIKV), cause neurological diseases. These diseases are due to viral infection of central nervous system (CNS) cells. The CNS is a privileged organ, isolated from pathogenic agents by a barrier between the blood and the barrier, called the blood-brain barrier (BBB). Neuropathogenic Flaviviruses which can cross this BBB in order to reach their target cells in the CNS, are neuroinvasive. This study aims at understanding the cellular mechanisms by which YFV and JEV Flaviviruses cross the BBB and the effects of viral infection by WNV and ZIKV of the CNS cells during neocortex development.YFV is a hepatrotopic virus, which mostly infects the liver and the kidneys. The two live-attenuated vaccines against YFV, the FNV (for French Neurotropic Virus) vaccine and the 17D vaccine, were obtained empirically by several passages in suckling mouse brain of YFV virulent strains. These vaccines do not cause any disease targeting the liver or the kidneys, but can sometimes cause post-vaccine encephalitis. These encephalitis cases suggest that the vaccine strains have become neurovirulent and neuroinvasive. Due to high risks of post-vaccine encephalitis, the FNV vaccine use was discontinued in the 1980s.JEV is a neurotropic virus, causing acute encephalitis in South East Asia. To date, there is a live-attenuated vaccine against JEV, the JEV SA14-14-2 vaccine, which was obtained empirically by several passages in primary hamster kidney cells. This vaccine is less neurovirulent and less neuroinvasive than JEV virulent strains in mouse model, and it protects against JEV infections. However, some cases of post-vaccine encephalitis were reported. It thus seems that, in some cases, the vaccine strain JEV SA14-14-2 is able to cross the BBB and infect neuronal cells.The recent ZIKV epidemics in French Polynesia and South America were linked to an increase in the number of congenital malformations, rising questions regarding the capacity of a Flavivirus to induce CNS congenital malformations.In this study, we have identified cellular mechanisms involved in Flavivirus neuroinvasion and studied the effect of ZIKV and WNV infection of neuronal cells under development.To study CNS development, we have infected mouse embryos brain slices. We were able to show that ZIKV has a preferential tropism for neuronal progenitors, whereas WNV has a preferential tropism for neuronal cells. We also show that infection of neuronal progenitors by ZIKV impairs the cell life cycle, whereas no effect on the cell life cycle was observed for WNV-infected cells. Studies on apoptosis induction did not show any difference between both viruses at early time points of infection.To study Flavivirus neuroinvasion, we have used an in vitro model of BBB composed of human endothelial hCMEC/D3 cells that can form tight junctions. These cells were cultivated on Transwell inserts and placed above human neuronal cells. Using this system, we show that YFV FNV cross the BBB more efficiently than YFV 17D, suggesting that YFV FNV is more neuroinvasive than YFV 17D. This observation can explain the higher post-vaccine encephalitis risks associated with YFV FNV vaccine compared to YFV 17D vaccine. We also confirmed that JEV SA14-14-2 vaccine strain is less neuroinvasive than JEV RP9.We also examined how JEV crosses the BBB and the endothelial cell response following JEV treatment. We show that both JEV RP9 and SA14-14-2 are able to cross the BBB without infecting its endothelial cells and without disrupting the BBB. Preliminary results suggest that JEV RP9, but not JEV SA14-14-2, crosses the BBB by dynamin-dependant transcytosis. Transcriptomic analysis of endothelial cells treated by either virus show slight, but significant, differences in regulation of genes implicated in several pathways associated with CNS diseases.

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.

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.