O desenvolvimento de uma vacina para o controle da dengue é uma prioridade em todo o mundo. O domínio helicase da proteína NS3 (NS3H) viral alberga epítopos reconhecidos por linfócitos T citotóxicos, os quais tem papel importante na eliminação de células infectadas. Esse trabalho propôs a obtenção de uma forma recombinante, produzida em linhagens de Escherichia coli, da NS3H do DENV2 com características similares à proteína nativa e sua utilização como um potencial antígeno vacinal. A NS3H foi obtida na forma solúvel, foi reconhecida por anticorpos de camundongos e de humanos infectados e foi capaz de interagir com o RNA viral. Camundongos imunizados com NS3H coadministrada com diferentes adjuvantes desenvolveram respostas imunológicas específicas mas não foram protegidos após desafio. Em conjunto, os resultados indicam que a proteína NS3H recombinante preserva conformação e determinantes antigênicos da proteína viral nativa e pode ser útil em estudos sobre a biologia viral e na busca de estratégias anti-virais voltadas para o controle da dengue. / The development of a dengue vaccine is a worldwide priority. The helicase domain of viral NS3 protein (NS3H) preserves epitopes recognized by cytotoxic T lymphocytes, which plays an important role in the elimination of infected cells. This study aimed the generation of a recombinant NS3H form of a type 2 dengue virus (DENV2) lineage, in Escherichia coli strains, with properties similar to the native protein and its use as a potential vaccine antigen. The NS3H was obtained in soluble form, was recognized by antibodies from mice and human subjects and was able to interact with the viral RNA. Mice immunized with NS3H combined with different adjuvants developed specific immune responses but did not confer protection to a lethal challenge. Altogether, the results indicate that the recombinant NS3H protein preserves conformational and antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV biology and the search for anti-virus approaches.

A dengue é uma doença infecciosa com grandes taxas de morbimortalidade, causada pelo vírus da dengue (DENV). Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas são infectadas anualmente em mais de 100 países tropicais e subtropicais de todos os continentes. O espectro clínico da infecção pelo DENV pode incluir formas assintomáticas ou sintomaticas que variam desde uma febre indeterminada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves denominados febre hemorrágica da dengue/síndrome do choque da dengue (FHD/SCD). Recentemente, ocorreu um dramático aumento do número de casos de FHD/SCD nas Américas, e este aumento coincidiu com a introdução do dengue sorotipo 3, genótipo III. No presente trabalho, objetivou-se a clonagem e a expressão das proteínas NS1 e NS3 do vírus da dengue tipo 3. As proteínas NS1 e NS3 do DENV-3 foram clonadas e expressas com sucesso em sistema procarioto. A amplificação dos genes das proteínas NS1 e NS3 foi realizada por RT-PCR, o qual gerou amplicons de cerca de 1050 e 1850 pb, respectivamente. Em seguida, os genes foram clonados por inserção dos amplicons no vetor plasmidial pCR-XL. Os genes de NS1 e NS3 foram subclonados no vetor de expressão pQE-30 através de sítios de restrição para as enzimas BamHI e HindIII. A expressão proteica foi obtida em sistema procarioto utilizando a cepa BL21(DE3) de E. coli, resultando em proteínas de 45 e 70 kDa as quais foram confirmadas por análises em Western blot utilizando como anticorpo primário fluido ascítico imune de camundongos e soro de pacientes com dengue. Estas proteínas virais podem ser utilizadas para estudos relacionados à patogênese, replicação e mecanismos de escape do sistema imune do DENV, além disso, podem ser potencias antígenos em métodos de diagnóstico. / Dengue is an infectious disease with high morbidity and mortality rates caused by dengue virus (DENV). According to the World Health Organization, about 50 to 100 million people are infected annually in more than 100 tropical and subtropical countries from all continents. The clinical spectrum of DENV infection can includes asymptomatic or symptomatic forms ranging from undetermined and self-limited fever, through dengue fever (DF) to severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Recently, there has been a dramatic increase in the number of cases of DHF/DSS in the Americas, and this increase coincided with the introduction of dengue virus type 3 (DENV-3), genotype III. The present study aimed to clone and express NS1 and NS3 proteins of DENV-3. The NS1 and NS3 proteins of DENV-3 was successfully cloned and expressed in a prokaryotic system. Amplification of NS1 and NS3 genes was carried out by RT-PCR, which yielded amplicons of approximately 1050 and 1850 bp, respectively. Then, the genes were cloned by inserting the amplicons into the plasmid vector pCR-XL. NS1 and NS3 genes were subcloned into the expression vector pQE-30 through the restriction sites for BamHI and HindIII enzymes. The protein expression was obtained in a prokaryotic system using the strain BL21 (DE3) of E. coli, resulting in 45 and 70 kDa proteins, which were confirmed by Western blot analysis using immune mouse ascitic fluid and serum of patients with dengue as primary antibody. These viral proteins can be used to study the pathogenesis, mechanisms of replication and immune escape of DENV, moreover, can be potential antigens in diagnostic methods.

La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes. / Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes.

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No. of bitstreams: 1 jerusabotelhosouza.pdf: 1803631 bytes, checksum: e5453b5a95de704649e79eb74037206d (MD5) Previous issue date: 2014-02-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A dengue é a arbovirose de maior importância para a saúde pública no Brasil. A cidade de Juiz de Fora vem passando por diversas epidemias de dengue nos últimos anos, com o registro de casos graves e óbitos. Diante deste contexto, este trabalho visou investigar a circulação de Dengue virus (DENV) em Aedes aegypti coletados do ambiente e em amostras clínicas de pacientes com suspeita de dengue por meio de técnicas moleculares e sorológicas. Amostras de Aedes aegypti (larvas e mosquitos) e soro de pacientes passaram pelo processo de extração de RNA total e este material foi utilizado para detecção de DENV por RT-PCR. Amostras clínicas de pacientes que apresentavam até seis dias de febre foram testadas para a proteína NS1 para Dengue virus. Além disso, foi realizada a análise das fichas dos pacientes nas quais foi possível verificar o dia de início da febre, os dados hematológicos (hematócrito, leucometria global e contagem de plaquetas) e sorológicos (IgM e/ou IgG). Aedes aegypti foi coletado em todas as regiões da cidade e dos 163 pools de larvas analisados, o DENV foi detectado em seis (cinco pools positivos para DENV-1 e um pool positivo para DENV-2). Estes pools haviam sido coletados nas regiões Norte, Centro e Sul. Das 166 amostras clínicas analisadas na detecção molecular, seis foram positivas (uma amostra positiva para DENV-1 e cinco amostras positivas para DENV-2). Sessenta amostras clínicas foram testadas para NS1 e 11 (18,33%) foram positivas. A partir da análise das fichas dos pacientes foi possível observar que 132 apresentavam resultados de sorologia. Destes, 39/132 (29,54%) apresentaram IgM, 9/132 (6,81%) apresentaram IgG e 15/132 (11,36%) apresentaram IgM e IgG. Além disso, 109 pacientes mostraram dados hematológicos. Entre estes, 18 (16,51%) pacientes apresentaram hematócrito inferior ao valor de referência, 32 (29,35%) desenvolveram leucometria abaixo de 3.500/mm3 e 24 (22,02%) tiveram contagem de plaquetas inferior ao valor de referência. Este é o primeiro estudo de investigação molecular de DENV em Juiz de Fora e os resultados indicaram a cocirculação dos sorotipos 1 e 2 em Juiz de Fora e a ocorrência da transmissão transovariana de DENV no Aedes aegypti. A combinação dos testes moleculares e sorológicos permitiu a identificação de 50 pacientes na fase aguda da doença e 22 na fase de convalescença. O conhecimento dos tipos virais circulantes no município e o diagnóstico da dengue em pacientes por mais de um teste e/ou parâmetro laboratorial constituem informações valiosas do ponto de vista epidemiológico e na estruturação de políticas públicas que visem o controle da dengue. / Dengue is the most important arboviral disease to public health in Brazil. The city of Juiz de Fora has undergone several dengue epidemics in recent years, with the record of severe cases and deaths. Given that, this work aimed to investigate the circulation of dengue virus (DENV) in Aedes aegypti collected from the natural environment and in clinical samples from patients with clinical symptoms of dengue, using molecular and serological approaches. Samples of Aedes aegypti (larvae and mosquitoes) and serum of patients were used for total RNA extraction, and the total RNA was used for detection of DENV by RT-PCR. Clinical samples from patients who had fever for up to six days were tested for Dengue virus NS1 protein. Furthermore, analysis of patient records was performed in which we could check the day the fever began the hematological data (hematocrit, total leukocyte count, and platelet count) and serologic response (IgM and/or IgG). Aedes aegypti was collected in all regions of the city and from the 163 larvae pools that were analyzed, DENV was detected in six (five pools DENV-1 positive and one pool DENV-2 positive). These pools were collected in the North, Centre and South regions. A total of 166 serum samples were analyzed for molecular detection, being six DENV positive (one serum sample was DENV-1 positive and five serum samples were DENV-2 positive). Sixty clinical samples were tested for NS1 and 11 (18.33%) were positive. From the analysis of patient records was possible to observe that 132 had serology results. Of these, 39/132 (29.54%) were IgM positive, 9/132 (6.81%) were IgG positive and 15/132 (11.36%) were IgM and IgG positive. In addition, from 109 patients hematological data were available. Among these, 18 (16.51%) patients had less than the reference value, 32 (29.35%) developed leukocyte hematocrit below 3.500/mm3 and 24 (22.02%) had platelet counts below the reference value. This is the first study of molecular investigation of DENV in Juiz de Fora, and the results indicated the co-circulation of serotypes 1 and 2 in Juiz de Fora and the occurrence of DENV transovarial transmission in Aedes aegypti. The combination of molecular and serological tests allowed the identification of 50 patients in the acute phase of the disease and 22 in the convalescent phase. The knowledge of viral types circulating in the municipality and the diagnosis of dengue in patients using different techniques constitute valuable information from an epidemiological point of view and help to structure public policies aiming the control of dengue.

<p>Flaviviruses are enveloped, positive-strand RNA viruses that are spread by mosquitoes and ticks and can cause serious disease in humans. Flavivirus virions undergo extensive structural changes during their life cycle, including during maturation and fusion. Flaviviruses are initially assembled at the endoplasmic reticulum in a non-infectious, immature state, and then traffic to the trans-Golgi network, where a pH drop triggers a structural rearrangement of glycoproteins prM and E on the virus surface from 60 trimers to 90 dimers. A host protease, furin, then cleaves prM which makes the transition irreversible. Upon exiting the host cell, pr disassociates from the virus and the infectious, mature virus is able to enter a new cell. <br></p><p><br></p> <p> </p> <p>In Chapter 1, an overview of flaviviruses is presented, including a brief history of their discovery and interaction with humans, followed by what is known about their life cycle and the maturation process. The structure of a mature flavivirus is then described, including the symmetrical arrangement of glycoproteins on the virion surface, the lipid membrane, and the nucleocapsid core, followed by an introduction of the structural proteins that assemble into the virion. The structure of the immature flavivirus is then described. The chapter concludes with a description of the dynamics and heterogeneity observed for flaviviruses.</p><p><br></p> <p> </p> <p>The conformational rearrangements that occur during flavivirus maturation remain unclear. The structures of immature and mature flaviviruses determined with cryo-electron microscopy (cryo-EM) demonstrated that flaviviruses are icosahedral particles with 180 copies of glycoproteins on their surface. Icosahedral viruses typically have a quasi-equivalent arrangement of glycoproteins, but flaviviruses lack quasi-equivalence and instead the three subunits within an asymmetric unit occupy different chemical environments. Although the subunits are the same proteins, the unique environment of each subunit can be exploited for tracking subunits during conformational rearrangements. For example, the unique labeling of a subunit can be used to identify it in the immature and mature virion.</p><p><br></p> <p> </p> <p>In Chapter 2, the maturation process was studied by developing tools to differentially label protein subunits and trap potential intermediates of maturation. The tools included heavy-atom compounds and antibody Fabs, which were used to probe Kunjin virus (KUNV), an Australian subtype of West Nile virus (WNV). One heavy-atom compound, potassium tetranitroplatinate(II), was found to derivatize immature KUNV, likely at sites on both E and prM. Higher-resolution studies will be required to determine if the compound differentially labeled the three subunits. The other tool developed was the E16 Fab. E16 Fab, originally isolated from a mouse immunized with WNV E and found to bind to two out of three subunits on mature WNV, was used to differentially label subunits in immature KUNV. Based on poor epitope accessibility on immature KUNV, E16 Fab was hypothesized to trap an intermediate state of maturation. In the cryo-EM reconstruction of E16 Fab bound to immature KUNV it was found that the virion had localized distorted density and apparent non-uniform binding of the E16 Fab. Based on this result it was proposed that flaviviruses had imperfect icosahedral symmetry. <br></p><p><br></p> <p> </p> <p>The structural asymmetry of immature and mature flaviviruses was investigated in Chapter 3. Icosahedral symmetry has always been imposed during cryo-EM reconstructions of flaviviruses, as it led to stable convergence of orientations. When reconstructions of immature KUNV and ZIKV were performed without imposing symmetry, the reconstructions showed that the flaviviruses had an eccentric nucleocapsid core, which was positioned closer to the membrane at one pole. At the opposite pole, the glycoprotein and inner leaflet densities were weak and distorted. Furthermore, there were protrusions from the core that contacted the transmembrane helices of the glycoproteins. In the asymmetric reconstruction of mature KUNV, the core was positioned concentric with the glycoprotein shell, in contrast to the immature virion, indicating that maturation alters the interactions between the core and the glycoproteins. The asymmetric reconstructions suggested that there is variable contact between the core and glycoproteins during assembly, which may be due to membrane curvature restrictions in the budding process. </p> <p> </p> <p><br></p><p>In Chapter 4, extracellular vesicles (EVs) that were released during dengue virus (DENV) infection were characterized by mass spectrometry. EVs may play a significant role in flavivirus infection, as they have been shown to transport both viral proteins and infectious RNA. EVs likely represent alternative modes of virus transmission and aid in immune evasion. However, previous studies on EVs are controversial because EVs are potential contaminated during assays by co-purifying virions and other particulates. The identification of EV biomarkers would greatly reduce contamination because biomarkers would enable isolation of pure EVs by affinity purification. Therefore, a strategy was developed to isolate EVs and profile them with proteomics. The four proteins cystatin-A, filamin B, fibrinogen beta chain, and endothelin converting enzyme 1 were found to be statistically enriched in the DENV sample and represent potential EV biomarkers. </p> <p> </p>

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>

<div> <div> <div> <p>Flaviviruses is a genus within the family Flaviviridae. The genus consists of more than 70 viruses, including important threatening human pathogens such as dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). These viruses are causative agents for a range of mild to lethal diseases and there are currently no US- licensed therapeutic treatments for infection. The virus genome is a positive-sense, single-stranded RNA, encoding ten viral proteins. Of the ten flavivirus proteins, Non- Structural protein 1 (NS1) remains the most elusive in terms of its functions. To date NS1 has been linked to disease pathology and progression and plays roles in virus replication and assembly. However, little is understood how NS1 orchestrates these functions and how NS1 from different viruses function distinctively from one another. Moreover, flavivirus NS1 has a peculiar ability to associate with lipid membranes. During the life cycle of NS1, the protein travels through the classical secretory path- way, similar to infectious virus particles, and is secreted into the extracellular space as mostly hexameric oligomers containing a lipid core. How the protein binds to lipids and whether such lipid binding is important for NS1 functions and overall flavivirus pathology remain unknown. Using structure-based mutagenesis, we found a group of mutants on WNV NS1, which particularly altered the viral specific infectivity but maintained wild-type level of virus replication. Purified mutated virus particles revealed that the specific infectivity alteration was not because of the particle but interaction of the virus particles and NS1 mutated proteins. Here we demonstrated that specific residues on NS1 were responsible for distinctly roles in NS1 functions and the virus specific infectivity was regulated by NS1 protein. In other structure-base study, we focused on the membrane association ability of NS1. All structure-predicted regions on NS1 were examined for its contribution for the membrane/lipid binding function. This interaction was required for NS1 biology activities including intracel- lular trafficking, oligomerization, and endocytosis. The lipidomes from deletion of each membrane association region revealed differences in lipid classes binding to each region and the composition flexiblity of the lipid cargo of NS1 hexamer. </p> </div> </div> </div>

The encompassing aim of this project was to gain a better understanding of the role of the cellular immune response to dengue virus (DV) infection. Dengue virus occurs as four distinct serotypes, called D1-D4. Symptomatic DV infection occurs as two forms of illness. The more severe form of DV infection, dengue hemorrhagic fever (DHF), is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. At its most severe, DHF can result in circulatory shock and death. Epidemiological studies indicate that DHF is more likely to occur following a secondary infection with a serotype of DV other than that which caused the primary infection, and there is evidence of increased T cell activation in more severe disease. These data and others indicate that DHF may be of an immunopathological nature. The memory CD4+ T cell response of a D4-immune donor was analyzed. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious DV antigens showed the highest proliferation to D4V antigen, with lesser, crossreactive proliferation to D2V antigen. CD4+ cytotoxic T lymphocyte (CTL) clones were established by stimulation with D4 antigen using a limiting dilution method. Seven out of 15 clones recognized the D4V capsid protein. The clones showed heterogeneity in their usage of T cell receptor Vα and Vβ genes. Six of these CTL clones were crossreactive between 02 and 04, and one clone was specific for D4. Using synthetic peptides, the D4V-specific clone was found to recognize an epitope between amino acids (aa) 47-55 of the capsid protein, while the crossreactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2 and D4. These results showed that the DV capsid protein can be a target of the cellular immune response following DV infection. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84-92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84-92. These results indicate that this donor exhibits memory CD4+ T cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level, but in bulk culture responses as well. Experiments were performed demonstrating that the CD4+ CTL clones were capable of mediating bystander lysis of non-antigen presenting target cells. Following activation on plate-bound anti-CD3 antibody or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Bystander lysis of neighboring, non-infected cells by activated CD4+ CTL clones might contribute to the pathology of DHF. The mechanisms of lysis employed by the T cell clones against both cognate and bystander target cells were assessed using chemical inhibitors of either the perforin- or Fas/FasL-mediated pathways. Three CD4+ CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/FasL interactions. In contrast, one clone used a Fas/FasL mechanism to lyse both cognate and bystander targets. These experiments indicated that the perforin- and FasL-mediated mechanisms of target cell lysis are not mutually exclusive, in that a single clone can kill target cells using either mechanism. Additionally, the ability of CD4+ CTL clones to lyse target cells by the perforin pathway indicates that, like CD8+ CTL, these clones might play a role in viral clearance and recovery from infection through lysis of virus-infected cells. Cytokine production by the capsid-specific CTL clones was also examined. Six of six clones studied produced high quantities of IFN-γ in response to either D2V antigen or the epitope peptide. IFN-γ was also produced by PBMC in a bulk culture from this donor stimulated with D4V antigen. All of the clones produced both TNF-α and TNF-β following stimulation. Four of six clones produced low amounts of IL-2, and only three of six clones produced detectable amounts of IL-4. Production of cytokines by activated CD4+ T cell clones in vivo could contribute to both viral clearance and immunopathology. To better understand the role that cytokine production might play in vivo in response to DV infection, cytokine mRNA levels were examined by PCR in DV-infected Thai children. mRNA for the cytokines IFN-γ, TNF-β, TNF-α, IL-1β, and IL-6 were detectable in the PBMC of DV-infected children. Semi-quantitative PCR analysis indicated that TNF-α mRNA levels were elevated in Thai children with DHF compared to children with classical dengue fever, the less severe form of illness (p=.013). All other cytokines showed no statistically significant difference between children with DHF and those with DF, although IFN-γ showed a trend toward elevation in more severe disease (p=.l). Increased production of TNF-α and/or IFN-γ in vivo could potentially contribute to the immunopathology of severe dengue illness. Taken as a whole, the data presented in this thesis provide a better understanding of the role of the cellular immune response to dengue virus infection and its potential contribution to the immunopathology of dengue hemorrhagic fever.

Dengue virus (DENV) causes over 500,000 infections annually with a spectrum of clinical diseases ranging from subclinical infection to dengue, a mild febrile illness, to life-threatening severe dengue. Vascular leakage without endothelial cell damage is the hallmark symptom of severe dengue illness and is proposed to be directly mediated by soluble inflammatory mediators IL-8 and TNFα. IL-8 production occurs in response to DENV infection, is elevated during severe dengue, is proposed to inhibit interferon, and could potentially recruit target cells to sites of infection. We previously showed that expression of DENV NS5 activates the IL-8 promoter, induces IL-8 transcription, and induces IL-8 protein production in HepG2 and HEK293A cell lines. As multiple DENV proteins are reported to interact with important signaling pathways, we hypothesized that other DENV proteins could contribute to the activation of IL-8. We found that plasmids expressing prM-E together, the GPI-linked variant of NS1 (NS1G), the carboxyl-terminal 112 amino acids of NS4B, as well as NS5 each induced expression from an IL-8 promoter-driven reporter plasmid. Expression of NS5 also induced activation of a RANTES promoter construct and TNFα mRNA expression. Further, we found that the carboxyl-terminal polymerase domain of NS5 was sufficient to induce IL-8 secretion but polymerase function was not required. Like NS5, prM-E and NS1G induced luciferase expression from an AP-1-driven reporter plasmid. We further tested whether activation of the IL-8 promoter depended on any single transcription factor within IL-8 using IL-8 promoter-driven plasmids mutated at the AP-1, C/EBP or NF-κB binding sites. We found that activation of the IL-8 promoter by prM-E, NS1G and NS4B did not depend on activation of any single transcription factor. Our data suggested that AP-1 may be both positively and negatively inducing transcription, fitting with previous theories that DENV regulates IL-8 induction. However, we did not observe any differences in activation of AP-1 subunit c-Jun, or the inhibitory subunits Fra-1 or Fra-2 between DENV and mock-infected cells. These data support a model in which multiple DENV proteins activate the IL-8 promoter, provide a potential basis of IL-8 induction by DENV in multiple cell types, and further supports a mechanism by which DENV contributes to severe dengue illness.

In Regionen, in denen verschiedene Flaviviren zirkulieren, wird die Diagnostik des West-Nil-Virus (WNV) oft durch Kreuzreaktionen erschwert. Einer dieser Staaten ist der Sudan, in welchem neben dem WNV auch Dengue-Viren (DENV) endemisch sind. Ziel dieser Arbeit war es, die WNV-Seroprävalenz im Sudan unter Ausschluss von Kreuzreaktionen mit Dengue-Viren oder dem Zika-Virus zu ermitteln. Zudem sollte bei Probanden mit früherer DENV-Infektion der Anteil an WNV-neutralisierenden Seren bestimmt werden. Serumproben von Patienten mit Fieber aus den sudanesischen Regionen Kassala, Nord-Kordofan und Red Sea State wurden vor Beginn dieser Arbeit mittels ELISA auf DENV-Antikörper untersucht. Seren ohne DENV-Antikörper (N = 106) und eine mit den Probanden dieser Gruppe hinsichtlich Herkunft, Alter und Geschlecht übereinstimmende zweite Personengruppe mit DENV-Antikörpern (N = 108) wurden ausgewählt. In allen Serumproben wurden mittels Mikroneutralisationstest sowohl die Häufigkeit WNV-neutralisierender Antikörper als auch deren Titer bestimmt. In der Gruppe der Seren ohne DENV-Antikörper neutralisierten 30,2% das WNV. Diese wurden in weiteren Neutralisationstests auf DENV- und ZIKV-neutralisierende Antikörper untersucht. 18,9% dieser Serumproben wiesen ausschließlich Antikörper gegen das WNV auf, sodass die ermittelte Seroprävalenz zwischen 18,9% und 30,2% lag. Die Seroprävalenz stieg mit zunehmendem Alter der Testpersonen an. Männer und Frauen waren gleichermaßen betroffen. Der Anteil der DENV-positiven Seren, die das WNV neutralisierten, betrug 83,3%. Diese Seren zeigten höhere WNV-Neutralisationstiter als die DENV-negativen Proben. Die Ergebnisse dieser Arbeit demonstrieren, dass ein erheblicher Anteil der Probanden eine serologisch nachgewiesene WNV-Infektion hatte. Im Vergleich dazu wies die große Mehrheit der Personen mit zurückliegender DENV-Infektion WNV-neutralisierende Antikörper auf. Es sind weitere Studien erforderlich, um klinische Fälle von WNV-Infektionen zu identifizieren und um herauszufinden, ob Personen mit kreuzneutralisierenden Antikörpern vor WNV-Infektionen geschützt sind.:Abkürzungsverzeichnis 1 Einführung 1.1 Charakterisierung, Verwandtschaft und Übertragung des West Nil Virus 1.2 Klinische Symptomatik einer West-Nil-Virus-Infektion 1.3 Verbreitung und Ausbrüche 1.4 Situation im Sudan 1.5 Serologische Diagnostik von West-Nil-Virus-Infektionen 2 Aufgabenstellung 3 Materialien und Methoden 3.1 Materialien 3.1.1 Chemikalien und Reagenzien 3.1.2 Enzyme und Farbstoffe 3.1.3 Nährmedien 3.1.4 Puffer 3.1.5 Antikörper 3.1.6 Kits 3.1.7 Virusstämme 3.1.8 Zellen 3.1.9 Patientenproben 3.1.10 Software und Programme 3.1.11 Technische Geräte und Materialien 3.2 Methoden 3.2.1 Kriterien zur Auswahl der Probanden 3.2.2 Zellkultivierung 3.2.3 Zellzählung 3.2.4 Mikroneutralisationstest 3.2.4.1 Screening 3.2.4.2 Antikörpertiter-Test 3.2.4.3 Interpretation 3.2.5 ELISA zum Nachweis Virus-infizierter Zellen 3.2.6 Bewertung der Ergebnisse 3.3 Statistik 4 Ergebnisse 4.1 Auswahl der Seren 4.2 Anteil der WNV-NT-positiven Probanden 4.3 Anteil der DENV- und ZIKV-NT-positiven Probanden 4.4 Vergleich der WNV-Seroprävalenz in Gruppe 1 und Gruppe 2 4.5 Charakteristika der WNV-NT-positiven Probanden 4.6 WNV-Antikörpertiter 5 Diskussion 5.1 Seroprävalenz des West-Nil-Virus 5.2 Assoziation zwischen zurückliegenden DENV-Infektionen und WNV-neutralisierenden Antikörpern 5.3 Häufigkeit von WNV-Infektionen in Kassala und Nord-Kordofan 5.4 Zusammenhang zwischen dem Alter und Geschlecht der Probanden und dem Anteil WNV-neutralisierender Antikörper 5.5 Methodenbeurteilung 5.6 Bedeutung der Untersuchungsergebnisse 6 Zusammenfassung der Arbeit 7 Literaturverzeichnis 8 Abbildungsverzeichnis 9 Tabellenverzeichnis 10 Formelverzeichnis 11 Selbstständigkeitserklärung 12 Lebenslauf 13 Danksagung