Dengue virus (DENV) infects 50-100 million people worldwide every year and is the causative agent of dengue fever (DF) and the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). There are four genetically and immunologically distinct DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Evidence suggests that an increased risk for DHF/DSS during secondary infection with a heterologous DENV serotype is due to an immunopathological response mediated by serotype-cross-reactive memory T cells from the primary infection. Furthermore, epidemiological studies have shown that the sequence of infection with different DENV serotypes affects disease severity. Though much has been learned from human studies, there exist uncontrollable variables that are intrinsic in this system such as genetic factors and unknown infection histories. These factors can skew experimental results, making interpretations difficult. Therefore, a murine model to study the immunologic aspects of sequential dengue infections would be an asset to the field of dengue research. To examine the effect of sequential infection with different DENV serotypes on the CD8+ T cell response, we immunized Balb/c mice with a primary DENV infection on day 0 and subsequently challenged with a heterologous secondary DENV infection on day 28. We tested all possible sequences of infection with the four serotypes. We analyzed the T cell response to two previously defined epitopes on the DENV E (Ld-restricted) and NS3 (Kd-restricted) proteins. Using ELISPOT and intracellular cytokine staining, we measured the frequency of T cells secreting IFNγ and TNFα in response to stimulation with these epitopes during three phases: acute primary, acute secondary, and the memory phase after primary infection. We found that the T cell response in heterologous secondary infections was higher in magnitude than the response in acute primary infection or during the memory phase. We also found that the hierarchy of epitope specific responses, as measured by IFNγ secretion, was influenced by the sequence of infections. The adoptive transfer of immune serum or immune splenocytes suggested that memory T cells from the primary infection responded to antigens from the secondary infection. In vitroexperiments with T cell lines generated from mice with primary and secondary DENV infections suggested the preferential expansion of crossreactive memory T cells. In testing all of the different possible sequences of infection, we observed that two different sequences of infection (e.g., DENV-2 followed by DENV-1 versus DENV-2 followed by DENV-3) resulted in differential CD8+ T cell responses to the NS3 peptide even though both secondary infection serotypes contain the identical peptide sequence. To investigate this phenomenon, we examined the role of CD4+ T cell help on the memory CD8+ T cell response. We found that CD4+ T cell cytokine responses differ depending on the sequence of infection. In addition, it was also shown that crossreactivities of the CD4+ T cell response are also sequence-dependent. Moreover, denguespecific memory CD4+ T cells can augment the secondary CD8+ T cell response. Taken together, we demonstrated that this serotype sequence-dependent phenomenon is the result of differential help provided by cross-reactive memory CD4+T cells. The findings in this novel mouse model support the hypothesis that both CD4+ and CD8+ serotype-cross-reactive memory T cells from a primary dengue virus infection alter the immune response during a heterologous secondary dengue virus infection. These data further elucidate potential mechanisms whereby the specific sequence of infection with different dengue virus serotypes influences disease outcomes in humans.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-15T13:37:53Z No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-19T12:59:17Z (GMT) No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) / Made available in DSpace on 2017-09-19T12:59:17Z (GMT). No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) Previous issue date: 2014-03-28 / CNPq / O gênero Flavivirus, família Flaviviridae, alberga arbovírus como os vírus dengue (DENV) e da febre amarela, que possuem importância médica e são envolvidos em epidemias de doença febril em áreas urbanas e rurais em regiões tropicais e subtropicais. No Brasil, atualmente, o DENV e o vírus da encefalite de Saint Louis (SLEV) são os dois flavivírus circulantes em áreas urbanas mais frequentes. Desde a introdução e emergência dos diferentes sorotipos de DENV a partir da década de 1980, extensas epidemias de febre do dengue vêm sendo reladas por todo o país. O SLEV, anteriormente reconhecido apenas em ciclos enzoóticos e com pouca relevância médica no Brasil, tem sido implicado em casos de doença febril durante epidemia de dengue no sudeste do país. O objetivo deste estudo foi investigar a circulação de flavivírus em pacientes com doença febril aguda com suspeita de dengue em Mato Grosso (MT) em 2011 e 2012. Material e Métodos: 604 amostras de soro obtidas entre outubro de 2011 e julho de 2012 de pacientes com doença febril aguda suspeita de dengue com até cinco dias do início dos sintomas em MT foram submetidas à multiplex semi-nested RT-PCR para a pesquisa de flavivírus, como os quatro sorotipos de DENV, SLEV, vírus da febre amarela, do Oeste do Nilo, Rocio, Bussuquara, Iguape e Ilhéus. Amostras positivas foram testadas em pelo menos duas reações independentes de single-nested RT-PCR e submetidas a sequenciamento nucleotídico de região do gene da glicoproteína de envelope para análise filogenética. Resultados: Dentre os 604 pacientes, 315 (52,2 %) foram positivos para DENV-4, 24 (4,0 %) para DENV-1, 3 (0,5 %) para SLEV, 1 (0,2 %) para DENV-2 e 1 (0,2 %) para DENV-3. Todas as amostras eram de pacientes oriundos de áreas urbanas de 17 municípios de MT. Entre as amostras positivas, 9 eram co-infecções entre DENV-1/DENV-4, 1 entre DENV-2/DENV-4, 2 por SLEV/DENV-4 e 1 entre SLEV/DENV-1/DENV-4. Os demais flavivírus pesquisados não foram detectados. Amostras negativas para flavivírus totalizaram 273/604 (45,20 %). Discussão: A ocorrência das arboviroses na população geralmente é subestimada, devido a fatores como quadro clínico inespecífico, infecções inaparentes e ausência de diagnóstico diferencial. O DENV-4 foi introduzido no MT em 2012, responsável pela maior casuística nas cidades da Baixada Cuiabana. Co-infecções são frequentes quando há circulação hiperendêmica dos quatro sorotipos do DENV, situação já relatada no Brasil em Manaus e Rio de Janeiro em 2011. O SLEV foi detectado em pacientes de Cuiabá e Várzea Grande. Infecções por SLEV são primariamente inaparentes ou brandas. Em MT, espécies de Culex e outros vetores deste virus são amplamente dispersas. Como humanos são hospedeiros finais e apresentam baixa viremia, sua ocorrência é provavelmente subestimada. Conclusão: DENV-1 e DENV-4 foram os flavivírus identificados com maior frequência. Os quatro sorotipos do DENV foram detectados em Cuiabá e infecções esporádicas pelo SLEV foram identificadas em pacientes co-infectados com o DENV-4 ou o DENV-4/DENV-1 em Cuiabá e Várzea Grande, indicando que outros arbovírus podem circular silenciosamente durante epidemia de dengue em áreas urbanas em MT. / The genus Flavivirus, Flaviviridae family, comprises arboviruses such as the medical important dengue virus (DENV) and yellow fever virus, involved in febrile illness epidemics in urban and rural areas of tropical and subtropical regions. In Brazil, DENV and Saint Louis encephalitis (SLEV) virus are currently the two most important flaviviruses circulating in urban areas. Since the introduction and emergence of different DENV serotypes in the 1980´s, extensive dengue outbreaks have been reported throughout the country.SLEV, previously recognized only in enzootic cycles without medical relevance in Brazil, has been implicated to febrile illness etiology during dengue fever outbreaks in the Southeast region. The aim of this study was to investigate the circulation of flaviviruses in patients with acute febrile illness suspected of harboring dengue in Mato Grosso (MT) between 2011 and 2012. Material and Methods: 604 serum samples obtained between October 2011 and July 2012 from patients with acute febrile illness suspected of dengue lasting less than 5 days in MT were subjected to multiplex semi-nested RT-PCR for flaviviruses, including all four serotypes of DENV, SLEV Yellow Fever, West Nile, Rocio, Bussuquara, Iguape and Ilheus viruses. Positive samples were tested at least twice in independent single-nested RT-PCR reactions and subjected to nucleotide sequencing of the envelope glycoprotein (E) gene region for phylogenetic analysis. Results: Among 604 patients, 315 (52.2 %) were positive for DENV-4, 24 (4.0 %) for DENV-1, three (0.5 %) for SLEV, one (0.2 %) for DENV-2 and one (0.2 %) for DENV-3. All patients are residents in urban areas of 17 cities of MT. Among then, 9 were co-infections among DENV-1/DENV-4, 1 between DENV-2/DENV-4, two between SLEV/DENV-4 and one with SLEV/DENV-1/DENV-4. The other flaviviruses were not detected. Negative samples for flavivirus totaled 273/604 (45.20 %). Discussion: The occurrence of arboviruses in the population generally is underestimated, probably due to unapparent infection or unspecific clinical presentation, associated to the absence of differential diagnosis. The DENV-4 serotype was introduced in MT in 2012, responsible for the largest number of cases in Cuiabá and Varzea Grande. Co-infections are common when hiperendemic circulation of all four serotypes of DENV is observed. This situation has already been reported in Brazil in Manaus and Rio de Janeiro cities in 2011. Three patients were positive for SLEV in Cuiaba and Várzea Grande. SLEV infections are primarily mild or unapparent. In MT, species of Culex and other vectors are widely dispersed. As humans are final hosts and, therefore, present low titer viremia, the occurrence of SLEV in the population is probably underestimated. Conclusion: DENV-1 and DENV-4 were the most frequently flaviviruses identified. The four DENV serotypes were detected in Cuiaba and sporadic SLEV infections were identified in patients co-infected with DENV-4 or DENV-1/DENV-4 in Cuiaba and Várzea Grande, indicating that other arboviruses may circulate silently during dengue epidemics in urban areas of MT.

Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste. / Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test

Dans le but de rechercher de nouveaux inhibiteurs de l’ARN polymérase NS5 du virus de la dengue (DENV), un criblage a été mené sur 650 plantes néo-calédoniennes. A la suite de ce criblage, deux espèces (Cryptocarya chartacea Kostermans et Trigonostemon cherrieri Veillon) ont été sélectionnées. L’extrait AcOEt des écorces de Cryptocarya chartacea (Lauraceae) a montré une forte inhibition de la NS5 polymérase (99 % à 10 µg/ml). L’étude phytochimique de l’extrait a mis en évidence une série de nouvelles flavanones 6-mono et 6,8-dialkylées, nommées chartacéones. Celles-ci sont présentes sous forme de mélanges racémiques au sein de C. chartacea. La chartacéone A a été purifiée sur colonne chirale conduisant à l’isolement de quatre diastéréoisomères optiquement purs. Une étude configurationnelle basée sur le calcul théorique de spectres de dichroïsme circulaire a permis la détermination de leur configuration absolue. Les chartacéones inhibent de façon sélective la NS5 polymérase du DENV. L’étude des extraits AcOEt des écorces et du bois de Trigonostemon cherrieri (Euphorbiaceae) a mis en évidence une série de métabolites secondaires originaux de type Diterpènes Daphnane Orthoester (DDO) chlorés : les trigocherrines (non-macrocycliques) et les trigocherriolides (macrocycliques). Ces composés ont montré une inhibition de l’activité enzymatique de la NS5 polymérase du DENV et une activité antivirale sur le virus du chikungunya in cellulo. / In order to identify new inhibitors of the dengue virus (DENV) NS5 RNA polymerase, a screening was led on 650 new-caledonian plants. Two species, Cryptocarya chartacea Kostermans and Trigonostemon cherrieri Veillon were selected. The EtOAc bark extract of Cryptocarya chartacea (Lauraceae) showed a potent inhibition of the NS5 polymerase activity (99 % at 10 µg/ml). The phytochemical study of the extract led to the isolation of a series of new 6-mono and 6,8-dialkylated flavanones, called chartaceones. Chartaceones are present as racemic mixtures in the plant. Chartaceone A was purified on chiral column leading to the isolation of 4 optically pure diastereoisomers. A configurational study based on the theoretical calculation of circular dichroism spectra allowed the determination of their absolute configuration. Chartaceone are selective inhibitors of the DENV NS5 polymerase. The study of the EtOAc extract from the bark and wood of Trigonostemon cherrieri (Euphorbiaceae) led to the isolation of a series of unusual chlorinated daphnane diterpene orthoesters (DDO) : trigocherrins (non macrocylic) and trigocherriolides (macrocyclic). These compounds inhibit the DENV NS5 polymerase activity and present an antiviral activity on the chikungunya virus in cellulo.

The immune response to viral infection involves a complexity of both innate and adaptive pathways at the cellular and the molecular level. There are many approaches to begin to define the pathways at work to control viral pathogenesis. The approach favored in this thesis was to conduct a broad screen of the innate immune response at the gene expression level of infected cells. The innate immune response is critical to the control of viral infections. Type I interferons (IFN), IFNα and IFNβ, are antiviral proteins that are an integral part of the innate immune response. Furthermore, by virtue of their effects on maturation and activation of antigen-presenting cells, IFNs are a pivotal link between the innate and adaptive immune systems. Most cell types produce type-I IFN when exposed to viruses. However, viruses have evolved multiple strategies to suppress IFN production or signaling. It is imperative to understand the virus-host interaction at the molecular level in order to identify as yet unknown mechanisms of the host antiviral response; these additional pathways may be useful in counteracting the viral suppression of IFN. Type-I IFNs regulate expression of at least five hundred genes, suggesting a complex network of signaling pathways. Depending on the cell type different proteins regulate the induction of IFN or the expression of IFN-inducible genes. Identification of proteins that induce selected IFN-inducible genes may provide synergistic activity with or may have an advantage over type-I IFN for anti-viral therapy in the future. Many diseases are untreatable if identified late in their progression. In resource-limited countries, many diseases are diagnosed clinically, which can lead to incorrect or delayed diagnosis and treatment. The identification of biomarkers of disease has the potential to guide the correct therapy in a timely fashion. The objective of this thesis was to identify novel anti-viral therapies and disease biomarkers for dengue virus (DENV) infection. DENV is a mosquito-borne positive-sense single-stranded RNA virus, which causes an estimated 50 million infections annually. Most DENV infections result in a febrile illness called Dengue fever (DF). Less frequently, infections cause Dengue hemorrhagic fever (DHF), a potentially fatal vascular leakage syndrome associated with the production of pro-inflammatory cytokines. At present patients infected with DENV can only be treated by intravenous fluid support to prevent hypovolemia and hypotensive shock. This treatment is less effective in severe cases if the diagnosis is delayed. Identification of therapeutics with both antiviral and immune-modulatory activity may lower patient mortality and reduce the burden of DENV on society. DENV infection is cleared in most individuals after a short period of viremia {Libraty, 2002 #2225}. Based on in vitro and mouse models, type-I and type-II IFN signaling pathways are thought to be critical in the regulation of DENV infection. Higher serum levels of type I and type II IFNs during acute DENV infection in patients lend support to the above hypothesis {Kurane, 1993 #2152; Libraty, 2002 #2225}. To understand the DENV-human host cell interaction at the molecular level, we performed global gene expression analysis on DENV-infected primary human cells using Affymetrix GeneChips (HG-U133A). We studied dendritic cells (DC), monocytes, B cells and human umbilical vein endothelial cells (HUVECs), all of which are known to be permissive to DENV infection. We first identified genes commonly regulated in multiple cell types in response to DENV infection; we hypothesized that understanding this common gene expression profile would identify signaling pathways involved in regulation of viral spread, activation of immune cells or induction of inflammation. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the 23 common response genes, was identified as a key link between type I and type II interferon response genes. Pretreatment of cells with recombinant TRAIL (rTRAIL) inhibited DENV replication in monocytes, B cells, HUVECs and DCs. Using the DC infection model, we showed that this inhibition of viral replication was apoptosis-independent. Type-I IFN receptor (IFNR) blocking experiments showed that signaling through the type-I IFN receptor played an important role in the antiviral activity of exogenous rTRAIL. Furthermore, TRAIL also significantly reduced the expression of mRNA and protein of pro-inflammatory cytokines (TNFα, MIP-1β and IFNα) and chemokines (MCP-2, IP-10 and IL-6) in response to DENV infection. The data that TRAIL inhibits both viral replication and pro-inflammatory cytokine production suggest that TRAIL has therapeutic value in dengue. The endothelial cell is the site of pathology in DENV infection in vivo (vascular permeability and plasma leakage). To understand the direct effect of DENV infection on endothelial cells and its role in the induction of genes regulating vascular permeability, we compared gene expression in DENV-infected HUVECs to that of uninfected cells and cells infected with other RNA and DNA viruses, including flaviviruses (West Nile, yellow fever, and Japanese encephalitis viruses), bunyaviruses (Sin Nombre and Hantaan viruses), Epstein-Barr virus and vaccinia virus. Among the genes confirmed for their differential expression, ST2 (Interkeukin-1 receptor-like-1 protein-IL1RL1) and indoleamine 2,3-dioxygenase (IDO) were identified to be upregulated specifically in response to DENV infection. Higher serum soluble ST2 (sST2) levels were detected in DENV-infected patients than in patients with other febrile illnesses (OFI) at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). In addition, patients with secondary DENV infections had higher serum sST2 levels compared with patients with primary DENV infections (p=0.047 at the last day of fever and p=0.030 at defervescence). Higher levels of IDO activity (pIn conclusion, global gene expression analysis identified novel proteins with promising characteristics for the treatment and/or diagnosis of DENV infection. Although further studies will be needed to validate the clinical utility of TRAIL, sST2, and IDO, these studies demonstrate the utility of this unbiased genomics approach to identify therapies to currently incurable diseases.

The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.

Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

Made available in DSpace on 2016-01-26T12:51:14Z (GMT). No. of bitstreams: 1 plinioluisluppino_tese.pdf: 1297185 bytes, checksum: 89ac3b7475f1bfd00a6808afd1ba661e (MD5) Previous issue date: 2007-06-04 / Dengue is the arthropod-borne transmitted viral disease of highest worldwide prevalence in mortality and morbidity. The proportion is pandemic ranging 1.6 million of infected patients yearly. Clinical presentation associated to epidemiological factors such as dengue prevalence in the patient s origin have been the only mean for early diagnosis. Laboratorial diagnosis, the conclusive, requires several days when there is viral isolation. Serological methods depend on high level of specific antibodies, and molecular methods are not available for the majority of laboratories of diagnosis and routine. The purpose of this study was to develop an agglutination method using latex to detect dengue virus, using biological samples of mice infected with dengue 1 Mochizuki strain by intracerebral via, and anti-dengue 1 specific antibodies from immunized mice. According to the results, this method was feasible for the dengue viruses diagnosis in positive samples of experimental animals. It provides further approaches for rapid detection of dengue in susceptible populations during the first days of the disease. / A dengue é a doença viral, transmitida por artrópode, de maior prevalência mundial em morbidade e mortalidade. Alcança proporções pandêmicas, estimando-se em 1,6 milhões de doentes anualmente. Manifestações clínicas características, associadas a fatores epidemiológicos, como prevalência da dengue na região de origem do paciente, têm sido os únicos instrumentos de diagnóstico precoce. O diagnóstico laboratorial, que é definitivo, demanda vários dias, quando realizado o isolamento viral. Métodos sorológicos dependem de níveis elevados de anticorpos específicos e os métodos moleculares não estão disponíveis para a maioria dos laboratórios de diagnóstico e rotina. Este estudo teve como objetivo desenvolver método de aglutinação do látex para a detecção do vírus dengue, utilizando amostras biológicas de camundongos infectados por via intracerebral com dengue 1, cepa Mochizuki e anticorpos específicos anti-dengue 1, obtidos de camundongos imunizados. Os resultados obtidos demonstraram a viabilidade deste método para diagnóstico do vírus Dengue em amostras positivas de animais de experimentação, abrindo novas perspectivas para o diagnóstico precoce da dengue na população susceptível, durante os primeiros dias de sintomas.

Made available in DSpace on 2014-07-29T15:26:24Z (GMT). No. of bitstreams: 1 tese valeria.pdf: 2053227 bytes, checksum: 9897e5628e288900b234c5acbe0fb42b (MD5) Previous issue date: 2008-08-06 / Nowadays, dengue constitute the major public health problem, because is relevant cause of illness and death between thousands people that resident in the tropical and subtropical regions in world. The dengue virus is classified as four serotypes (DENV-1, 2, 3 and 4) according to antigenic differences and characterized intra-typical groups called genotypes. The laboratorial surveillance enables the diagnostic confirmation of dengue infection and monitoring serotypes circulating through the routine diagnostic techniques. Recently, the use molecular techniques has contributed to characterize and monitoring of the genotypes potentially virulent during epidemic and knowledge of biology of dengue virus. This thesis was organized in an introduction section, that include a literature review on dengue, and two manuscripts that describing the research conducted with focus on laboratory diagnostic and molecular epidemiology. The first manuscript entitled Laboratorial Surveillance of Dengue Virus in Central-Brazil, 1994-2003, was published at Journal of Clinical Virology, 2006 37 (3): 179-83. In this study we present the results of the virological surveillance for dengue cases conducted in the city of Goiânia (~1,200,000 population) from 1994 to 2003. Suspected cases were from the main public infectious disease reference hospital and outpatient clinics covering the metropolitan area. Serological and virus isolation tests were conduced at the regional reference laboratory. Our objective was to report dengue circulating serotypes from 1994 to 2003 and the role of distinct serotypes on dengue clinical outcomes in Central Brazil and to characterize serotypes and genotypes by reverse transcriptase PCR (RT-PCR) and by restricted site-specific PCR (RSS-PCR) patterns in selected samples. Laboratory surveillance identified mainly DEN-1 serotype from 1994 to 2002 shifting to a high circulation of DEN-3 in 2003. The adults (87,4%) were the most affected group and dengue fever accounted for the majority of the cases. Diagnosis of dengue was confirmed in ~50% of the suspected and enhanced by RT-PCR. RSS-PCR patterns for DEN-1 and DEN-3 corresponded to the circulating subtypes in the country. The infection DENV-3 did not suggest a major role of infecting DEN-3 in increasing disease severity during its first-year spread in Central Brazil. The second manuscript, to be submitted for publication is entitled: Epidemiologia Molecular do Vírus Dengue tipo 3 em Goiânia GO, 2005-2006. The objective of this manuscript was to characterize the DENV-3 genotype circulating isolated from well-characterized clinical and laboratory samples in Goiânia-GO/Brasil. Seven samples had sequences of the prM/M/E region obtained and comparative analysis was performed with the reference strains. The results showed the homology of the genomics sequences with genotype III strains. The nucleotide identity of the all the samples varied from 97.0% to 99.6% and the amino acid sequences from 97.5% to 99.5%. The analysis of the nucleotide sequence revealed silent mutation and 14 amino acid changes in the protein deduced from gene prM/M/E. In conclusion, the study confirms that the strains of DENV-3 in Central Brazil relate to genotype III. The genomic changes along Domain III of the protein E were observed, which could affect the pathogenicity, but were not consistent between samples of DCC and DHF. Samples of patients with dengue fever had mutations related to viral attenuation. More investigation is necessary to evidence of genomic changes found in relationship with clinical forms. / Atualmente, a dengue representa um dos maiores problemas em saúde pública, pois é causa de doença e morte entre milhares de pessoas que residem nas regiões tropicais e subtropicais do mundo. Os vírus dengue existem como quatro sorotipos virais (DENV-1 a DENV-4) de acordo com diferenças antigênicas, sendo caracterizados grupos intra-típicos denominados genótipos. A vigilância laboratorial possibilita a confirmação diagnóstica de infecção por dengue e o monitoramento de sorotipos circulantes, através de técnicas diagnósticas de rotina. Recentemente, o uso de técnicas moleculares tem contribuído para a caracterização e monitoramento de genótipos potencialmente virulentos na vigência de epidemias e melhor entendimento da biologia do vírus. Esta tese de doutorado compreende uma introdução que contempla uma revisão da literatura sobre dengue e dois manuscritos que descrevem as pesquisas realizadas com enfoque no diagnóstico laboratorial e epidemiologia molecular. O primeiro manuscrito intitulado Laboratorial Surveillance of Dengue Virus in Central-Brazil, 1994-2003, foi publicado no Journal of Clinical Virology, 2006 37 (3): 179-83. Neste estudo, apresentamos os resultados de uma vigilância laboratorial conduzida na cidade de Goiânia na região Centro-Oeste do Brasil, desde a introdução dos vírus dengue em 1994 até 2003. Casos com suspeita clínica de dengue foram atendidos em unidades de saúde e hospitais de referência da região metropolitana e os testes sorológicos e/ou isolamento viral realizados no laboratório de referência estadual LACEN-GO. Nosso objetivo foi descrever sobre os sorotipos virais circulantes de dengue nos anos de 1994 a 2003 e o seu papel nos desfechos clínicos de dengue na região Centro-Oeste do Brasil e caracterizar sorotipos e genótipos ou subtipos genômicos pela RT-PCR e RSS-PCR em amostras selecionadas. O sorotipo DENV-1 foi identificado como prevalente nos anos 1994 a 2002, sendo substituído por DENV-3 em 2003. Os adultos (87,4%) foram mais atingidos e a dengue clássica a mais diagnosticada. A dengue foi confirmada em 50% dos casos suspeitos de dengue incrementado pela RT-PCR. Os genótipos ou subtipos genômicos do DENV-1 e DENV-3 corresponderam circulantes no país. A infecção pelo DENV-3 não sugere um papel relevante no aumento da gravidade da doença durante seu primeiro ano de dispersão no Centro-Oeste do Brasil. O segundo manuscrito, a ser submetido à publicação, foi intitulado Epidemiologia Molecular do Vírus Dengue tipo 3 em Goiânia GO, 2005-2006. O objetivo desse trabalho foi caracterizar o genótipo circulante do DENV-3 isolado de amostras bem caracterizadas clínica e laboratorialmente em Goiânia-GO/Brasil e analisar as alterações genômicas encontradas. Sete seqüências regiões prM/M/E, foram obtidas e analisadas em comparação com cepas protótipo do DENV-3. As seqüências do estudo apresentaram maior homologia com a cepas do genótipo III. O percentual de identidade nucleotídica das amostras do DENV-3 seqüenciadas variou de 97,0% a 99,6% e de aminoácidos 97,5% a 99,5%, mostrando-se conservadas. A análise da seqüência nucleotídica das amostras do estudo revelou mutações silenciosas e 14 substituições de aminoácidos na seqüência da proteína deduzida do gene prM/M/E. Em conclusão, o estudo confirma que as cepas de DENV-3 da região centrooeste pertencem ao genótipo III. Alterações genômicas significativas foram observadas ao longo do domínio III da proteína E, as quais poderiam afetar a patogenicidade, entretanto não foram consistentes entre as amostras de DCC e FHD. As amostras de pacientes com dengue clássica apresentaram alterações em resíduos de aminoácidos no domínio III relatados como pontos de atenuação viral. Maiores investigações serão necessárias para confirmar as alterações encontradas e sua relação com as formas clínicas.

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.