Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells.

FSME-spezifische Immunität bei FSME-geimpften Kindern und Jugendlichen mit JIA Die juvenile idiopathische Arthritis (JIA) ist eine chronische entzündliche Autoimmunerkrankung des Kindesalters mit unbekannter Ätiologie. Die standardmäßigen Impfungen sind gemäß der aktuellen STIKO-Empfehlungen empfohlen. Die Frühsommer-Meningoencephalitis (FSME) ist eine durch Zecken übertragene Krankheit, die zu Fieber und neurologischen Symptomen führen kann. Eine kausale Therapie gibt es nicht, eine Impfung gegen FSME ist allerdings für Personen empfohlen, die in Risikogebieten leben oder diese bereisen. Die vorliegenden Untersuchungen der IgG-anti-FSME-Konzentration und –Avidität, des Neutralisationstiters und des INF γ-ELISPOTs umfassen die Daten von 100 JIA Patienten und 31 gesunden Kontrollen und überprüfen, ob die humorale und zelluläre Immunität gegen FSME von Patienten mit JIA vergleichbar mit der von gesunden Kontrollen ist. Die Marker der humoralen Immunität waren vergleichbar zwischen den zwei untersuchten Gruppen. Sowohl bei der IgG-anti-FSME-Konzentration und –Avidität als auch bei den Neutralisationstests waren die Mittelwerte beziehungsweise der Median bei den Patienten mit JIA niedriger verglichen mit den gesunden Kontrollprobanden. Bei den Ergebnissen des INF γ-ELISPOTs war es umgekehrt, aber niedrige humorale Parameter korrelierten nicht mit niedrigen Parametern der zellulären Immunität oder andersherum. Entsprechend der INF γ-ELISPOT Ergebnisse wurden die Patienten mit JIA und die gesunden Kontrollen in High, Low und Non Responder aufgeteilt. Prozentual gab es bei den gesunden Kontrollprobanden mehr Low und Non Responder. Insgesamt gab es keine statistisch relevante Korrelation zwischen der humoralen und zellulären Immunität. Die Anzahl der Impfungen und auch Anzahl der Boosterimpfungen nach der Grundimmunisierung hatten eine bedeutende positive Auswirkung besonders auf neutralisierende Antikörper bei Patienten mit JIA und aber auch auf die IgG-anti-FSME-Konzentration und –Avidität sowie die zelluläre Immunität bei JIA Patienten und gesunde Kontrollen. Eine Therapie mit TNF α-Inhibitoren beeinflusste den Neutralisationstiter bei den Patienten mit JIA signifikant negativ, wobei sich eine Therapie mit MTX signifikant negativ auf die IgG-anti-FSME-Avidität auswirkte. Als Schlussfolgerung lässt sich sagen, dass die Impfung gegen FSME ebenso wirksam bei den Patienten mit JIA wie bei gesunden Kindern und Jugendlichen ist, Boosterimpfungen bei Patienten mit JIA aber sehr wichtig sind und öfters empfohlen werden sollten. / TBE-specific immunity in TBE vaccinated children and adolescents with JIA Juvenile idiopathic arthritis (JIA) is a chronic inflammatory autoimmune disease in childhood with unknown etiology. The standard vaccinations are recommended in accordance with the current STIKO recommendations. Tick-borne encephalitis (TBE) is an Illness that can lead to fever and neurological symptoms. There is no therapy, but a vaccination against TBE is recommended for individuals who live in or travel through high-risk areas. The present experiments of IgG-anti-TBE-concentration and -avidity, of the neutralization titer and the INF γ-ELISPOT include the data of 100 JIA patients and 31 healthy controls and examine whether the humoral and cellular immunity against TBE in patients with JIA is comparable to the one of healthy controls. The markers of humoral immunity were comparable between the two groups. The mean values and the median of the IgG-anti-TBE-concentration and -avidity as well as the neutralization tests, were lower in the group of patients with JIA compared to the healthy control subjects. In the results of the INF γ-ELISPOT it was the other way around, but low humoral parameters did not correlate with low parameters of cellular immunity or vice versa. According to the results of the INF γ-ELISPOT patients with JIA and healthy controls were divided into high, low and non-responders. Within the healthy control group there were more low and non-responders. In total there was no statistically relevant correlation between humoral and cellular immunity. The number of vaccinations and also the number of booster vaccinations after basic immunization had a significant positive impact especially on neutralizing antibodies in patients with JIA and also to the IgG anti-TBE concentration avidity and cellular immunity in JIA patients and healthy controls. A therapy with TNF α inhibitors had a significant negative influence on the neutralization titer in patients with JIA whereas the therapy with MTX had a significant negative effect on IgG anti-TBE avidity. In conclusion, it can be said that vaccination against TBE is equally effective in patients with JIA as in healthy children and adolescents, that booster vaccinations for patients with JIA are very important and often recommended should be.

West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic. / Master of Science / West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging flaviviruses transmitted by mosquito bites, primarily among perching birds. However, mosquitoes can also transmit these viruses to animals and humans, especially in regions where these viruses are prevalent. The immune system, which defends against pathogens and other diseases, usually combats these viruses effectively, preventing most people from developing symptoms. The immune system has two main branches: the innate immune system, which confers immediate defense, and the adaptive immune system that includes antibodies and certain long-lasting memory cells, that can fight off infections years after the initial exposure to the same or similar disease-causing agents. Occasionally, the immune system fails to fight these viruses, particularly in the elderly or those with chronic diseases, leading to fever or severe brain inflammation called encephalitis. Currently, WNV and SLEV are circulating in the Americas, while WNV and USUV are present in European countries. Due to similar transmission methods, infection patterns, and geographical overlap, individuals might be sequentially infected with WNV and USUV in Europe, and WNV and SLEV in the Americas in their lifetime. These viruses also share common antigens, which can induce similar immune responses. Therefore, the immune response to one virus might protect against another with similar antigens. It has been reported that the immune response induced by WNV can protect against encephalitis caused by USUV or SLEV. However, it remains unclear whether this cross-protection is mediated by antibodies or a certain type of immune cells called T cells. This study investigates whether antibodies induced by WNV infection can protect against SLEV or USUV in a mouse model. Plasma, the part of blood containing antibodies, is referred to as convalescent plasma when collected after an individual has recovered from an infection or disease. Human WNV convalescent plasma was tested against SLEV and USUV using a plaque reduction neutralization test to determine the antibodies’ ability to prevent viral infection in a laboratory setting. Human WNV convalescent plasma effectively prevented SLEV and USUV from infecting cells. We then developed a mouse model that could be infected with SLEV or USUV and mimic human disease. Groups of mice were systematically transferred with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum one day before the infection with WNV, SLEV, or USUV. Disease conditions, such as weight loss, reduced movement, hunchback, fur loss, and occasional paralysis, were monitored until the infected mice were humanely euthanized. After euthanasia, the brains of the mice were collected to measure viral load and examine signs of encephalitis. We observed asymptomatic disease outcomes reflecting natural human infection. Both human and mouse WNV convalescent samples reduced viral load in the blood for a period in both SLEV and USUV-challenged groups. Mice treated with human WNV convalescent plasma showed a trend of lower SLEV in their brains. Additionally, mice treated with mouse WNV convalescent serum had lower SLEV titers in their brains compared to those treated with mouse normal serum. Overall, these findings suggest that human WNV convalescent plasma provides some crossprotection against SLEV- and USUV-induced diseases. Understanding the mechanism of crossprotection is crucial for developing therapeutics against these viruses and predicting disease outcomes in areas where multiple viruses of the Japanese encephalitis virus serocomplex are prevalent.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. The main symptoms of PD include tremor, muscle stiffness, slowness of movement, and postural instability. Motor symptoms of PD are driven by pathology primarily in the substantia nigra (SN) region of the brain, where there is dopaminergic neuron death and a-Synuclein (a-Syn) protein aggregation. Simultaneously, there is persistent inflammation in the central nervous system (CNS) and in the periphery of PD patients that begins prior to the onset of motor symptoms. Animal models of PD can exhibit inflammation in the brain and periphery, but the role of the immune system in disease pathogenesis and progression is not fully understood. In Chapter 1, I introduce PD neuropathology, genetic and environmental risk factors, and hypotheses for a-Syn aggregation and propagation from the periphery to the brain. Then, I discuss the altered immune responses and features of autoimmunity in PD patients. Lastly, I describe animal models of PD and how they are used to investigate the role of the immune system in PD pathology. Features of autoimmunity against the a-Syn protein are present in PD patients and appear prior to the onset of motor symptoms. Whether autoimmunity to a-Syn can alone initiate PD pathology has not been tested in animal models of PD. In Chapter 3, I demonstrate that activation of a-Syn32-46-specific CD4+ T cells in transgenic humanized mice induced early features of PD in the gut. We found that humanized mice immunized with a-Syn32-46 experience constipation, a common early PD symptom, as well as enteric neuron loss. Further investigation into the immune response in the gut revealed activated innate and adaptive immune responses which promoted dopaminergic neuron loss in the small intestines. PD pathology, however, did not develop in the brain, suggesting that autoimmunity to a-Syn32-46 promotes pathology at prodromal PD stages. Elevated T cell infiltration is found in the PD patient brain, suggesting that the adaptive immune response may also promote pathology in the brain at later disease stages. I hypothesized that a-Syn-specific CD4+ T cells may promote PD neuropathology upon a-Syn aggregation. In Chapter 4, I tested this hypothesis in both humanized and wild-type mice. I found that active immunization with a-Syn32-46 in humanized mice combined with human a-Syn overexpression in the brain did not increase T cell CNS infiltration. However, adoptive transfer of a-Syn-specific T cells into wildtype mice that overexpress human a-Syn increases T cell CNS infiltration and CNS myeloid cell activation. These results suggest that a-Syn-specific T cells may promote neuroinflammation in the presence of a-Syn accumulation. In Chapter 5, I summarize the major findings from my research and future directions which can provide a more detailed understanding of the ways that a-Syn-specific T cells promote PD neuropathology. Furthermore, I discuss innovations in the diagnosis and treatment of PD and the potential for immunotherapies to significantly modify disease course.

Bacterial meningitis in dogs and cats is a rare disease associated with a high lethality rate. The spectrum of causative bacteria includes a diverse set of gram positive, gram negative and anaerobic species. Currently, no veterinary medicinal product is approved for this indication in these species in Europe. The objective of this review was to collect the available pharmacokinetic data for antibiotics approved in dogs and cats to enable a preliminary analysis of their potential effectiveness for the treatment of bacterial meningitis. This analysis yielded data for 13 different antibiotics in dogs and two in cats. Additionally, data about frequently recommended cephalosporines not approved in dogs and cats were included. The collected data was used to assess the potential of the respective antibiotics to attain certain simple pharmacokinetic-pharmacodynamic (PK-PD) indexes in the cerebrospinal fluid (CSF). A more sophisticated investigation using modern methods was not possible due to the limited data available. For this purpose, data about the sensitivity of four bacterial species commonly associated with meningitis in dogs and cats to these antibiotics were included. The analysis provided evidence for the potential effectiveness of ampicillin, doxycycline, enrofloxacin, ceftriaxone and cefoxitin against bacteria frequently detected in bacterial meningitis in dogs. Data were not available or insufficient for the assessment of several antibiotics, including frequently recommended substances like metronidazole and trimethoprimsulphonamide. Little evidence is available for the use of antibiotics in cats afflicted with this disease, highlighting the need for further research to obtain data for evidence based therapeutic recommendations.

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

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.

The main research problem concerns the characteristics of tick-borne disease and determine the quantity and quality of information that students get in health education and biology of these diseases. The theoretical part deals with the history, etiology, clinical features, diagnosis, treatment and prevention of tick-borne encephalitis and Lyme disease. It also discusses the general problems of tick and other diseases that ticks carry and cause. Also is mentioned the inclusion of tick-borne diseases in the educational program. The practical part is devoted to the awareness of upper primary school students and changes in their knowledge about the issue during their elementary education. The aim of this thesis is to determine how the students are informed about diseases transmitted by ticks, especially about Lyme disease and tick-borne encephalitis. This method is used questionnaire, which includes questions regarding this issue. Although the results of the research show a certain degree of knowledge of students at upper primary school, especially in some classes is unsatisfactory. Based on research, there is provided a recommendation for pedagogical practices. KEYWORDS: tick, tick-borne encephalitis, Lyme disease, knowledge, disease, upper primary school

INTRODUÇÃO: A encefalite de Rasmussen (ER) é uma doença rara e esporádica, apresentando-se como uma síndrome com disfunção cerebral multifocal e convulsões focais refratárias ao tratamento medicamentoso, por vezes se manifestando com epilepsia parcial contínua. O início das crises focais predomina na infância e afeta crianças previamente hígidas, com curso progressivo. Na maioria das vezes envolve apenas um hemisfério cerebral, que se torna atrófico. O diagnóstico é feito através da análise do eletroencefalograma, características clínicas, achados de ressonância magnética (RM) e/ou achados histopatológicos. A RM encefálica é particularmente útil no estudo destes doentes, fornecendo dados que podem contribuir para o diagnóstico, ajudar na seleção do local para biópsia, assim como no acompanhamento da evolução progressiva da doença. MATERIAIS E MÉTODOS: Foram coletados os dados demográficos, avaliações neuropsicológicas, dados cirúrgicos e achados de imagem de todos os pacientes diagnosticados com ER no HCFMRP, de 1997-2016. RESULTADOS: Foram incluídos 35 pacientes com média de idade de 5,8 anos. Setenta por cento destes apresentaram-se com epilepsia parcial contínua e 29 tiveram também a confirmação histopatológica. Não houve nenhum caso de acometimento bilateral confirmado nesta amostra. Os achados de imagem mais comuns foram alteração de sinal, atrofia focal ou hemisférica e dilatação ventricular, em graus variados. Trinta e três pacientes foram submetidos ao tratamento cirúrgico. CONCLUSÕES: A definição da conduta e tratamento dos pacientes com ER deve ser discutida por equipe multidisciplinar, levando em consideração os achados clínicos, EEG e de exames de imagem, com objetivo de controlar as crises a fim de minimizar os déficits cognitivos, motores e de linguagem destes pacientes. / INTRODUCTION: Rasmussen encephalitis (RE) is a rare and sporadic disease, presenting as a multifocal brain dysfunction and focal seizures, refractory to drug treatment, sometimes manifesting with continuous partial epilepsy. The onset of seizures predominates in childhood, and affects previously healthy children with progressive course. Most often involves one cerebral hemisphere, which becomes atrophic. Diagnosis is made through EEG analysis, clinical, magnetic resonance imaging (MRI) findings and/or histopathological findings. Brain MRI is particularly useful, and provides data that can contribute to the diagnosis, help in site selection for biopsy, as well as in monitoring the progressive course of the disease. MATERIALS AND METHODS: We collected demographic data, neuropsychological evaluations, surgical and imaging findings of all patients diagnosed with RE in HCFMRP, from 1997-2016. RESULTS: It included 35 patients with a mean age of 5.8 years. Seventy percent of these presented with continuous partial epilepsy and 29 also had histopathologic confirmation. There were no cases of confirmed bilateral involvement in this sample. The most common imaging findings were signal change , focal or hemispheric atrophy and ventricular dilatation , in varying degrees. Thirtythree patients underwent surgical treatment. CONCLUSIONS: The definition of management and treatment of patients with ER should be discussed by a multidisciplinary team, taking into account the clinical, EEG and imaging findings, in order to control seizures and minimize cognitive, motor and language deficits of these patients.