O vírus do Oeste do Nilo (VON) é um Flavivírus capaz de infectar muitas espécies de vertebrados, incluindo o homem. Embora reconhecida desde 1940, esta virose nunca havia sido descrita nas Américas, onde emergiu nos Estados Unidos ao final da década de 1990, com numerosos casos de meningoencefalite em humanos. Posteriormente, sua transmissão por transfusão de sangue e órgãos foi comprovada, levando à implantação de testes moleculares (NAT) para a triagem de doadores nos EUA e Canadá a partir de 2003. Nos anos seguintes, o VON foi sendo progressivamente detectado em países como México, Panamá e áreas do Caribe, sugerindo sua iminente introdução na América do Sul. De fato, evidências sorológicas foram reveladas em cavalos e aves na Colômbia, Venezuela, Argentina e muito recentemente no pantanal mato-grossense (em cavalos). A vigilância epidemiológica para este agente é de grande importância para a saúde pública, visto o potencial de morbimortalidade deste vírus para humanos. Sendo assim este trabalho tem o objetivo de investigar a presença do RNA do VON em amostras de doadores de sangue, pacientes com meningoencefalite ou febre de origem indeterminada e soros e amostras cerebrais de equinos. Foram analisadas 2.202 doações de sangue do Amazonas (HEMOAM), 3.144 do Mato Grosso do Sul (HEMOSUL); líquido cefalorraquidiano de 51 pacientes com suspeita de meningoencefalite viral (Hospital das Clínicas/FMUSP, São Paulo) e soro de 198 pacientes com síndrome febril aguda, negativos para Dengue e Malária (Fundação de Medicina Tropical de Manaus). Além disto, 293 amostras de soros de equinos da região do Pantanal e 63 biópsias de tecido cerebral de cavalos que foram a óbito por encefalite de etiologia desconhecida. Estas amostras foram submetidas ao teste automatizado cobas TaqScreen WNV (Roche) na plataforma cobas s201 em sistema de pool de 6 unidades (doações de sangue) ou individualmente (pacientes). Todas as amostras apresentaram amplificação satisfatória do controle da reação, porém nenhuma apresentou resultado positivo para a presença do RNA do VON. Embora já exista evidência da exposição de equinos no Brasil ao VON, não parece haver até o momento, disseminação importante deste agente entre humanos e equinos, uma vez que o RNA viral não foi detectado nem em doadores de sangue e nem em equinos, incluindo os de cidades próximas aos locais onde cavalos soropositivos foram encontrados (Corumbá MS). / The West Nile Virus (WNV) is a Flavivirus able to infect many species of vertebrates, including man. Recognized since 1940, this virus had never been described in the Americas, which emerged in the United States at the end of the 1990s, with numerous cases of meningoencephalitis in humans. Later, transmission by transfusion of blood and organs was confirmed, leading to the deployment of molecular testing (NAT) for screening of donors in the U.S. and Canada since 2003. In the following years, WNV has been progressively detected in countries like Mexico, Panama and the Caribbean areas, suggesting their imminent introduction in South America In fact, serological evidence was revealed in horses and birds in Colombia, Venezuela and Argentina and most recently in Pantanal, Mato Grosso (horses). Epidemiological surveillance for this agent is of great importance to public health, given the potential morbidity and mortality of this virus to humans. Therefore this study aims to investigate the presence of WNV RNA in samples of blood donors, patients with meningoencephalitis or fever of unknown origin and serum and brain samples from horses. We analyzed 2202 blood donations from Amazon (HEMOAM), 3144 from Mato Grosso do Sul (HEMOSUL); cerebrospinal fluid of 51 patients with suspected viral encephalitis (Hospital das Clínicas / FMUSP, São Paulo) and serum samples from 198 patients with acute febrile syndrome, negative for Dengue and malaria (Foundation for Tropical Medicine in Manaus). In addition, more 293 serum samples from horses of the Pantanal and 63 biopsies of brain tissue from horses that died of encephalitis of unknown etiology. These samples were subjected to automated cobas TaqScreen WNV test (Roche) on the platform in cobas S201with a system of 6 units pool (blood donations) or individually (patients). All samples showed satisfactory control amplification, but none showed as positive for the presence of RNA VON. Although there is already evidence in horses in Brazil of exposure to WNV, there seems to be far that an important spread of this agent between humans and horses, since the viral RNA was not detected either in blood donors or in horses, including cities near the locations where seropositive horses were found (Corumbá - MS).

Made available in DSpace on 2014-07-29T15:07:28Z (GMT). No. of bitstreams: 1 dissertacao FINAL RICARDO.pdf: 485243 bytes, checksum: 1906f1911ad5edaec8d73c8546aaca3e (MD5) Previous issue date: 2010-07-02 / The arboviruses are diseases that affect humans, horses and other animal species causing in the majority of cases an asymptomatic infection to neurological disorders. The Flaviviruses are important arbovirus found in Brazil. A descriptive study was conducted from serological surveys in 1775 horses for detection of anti-Flavivirus antibodies in the State of Paraná (Foz do Iguaçú, Maringá and Barracão), Santa Catarina (São Miguel do Oeste, Guaraciaba, Iraceminha, Dionisio Cerqueira, Guarujá do Sul and São José do Cedro) and Rio Grande do Sul (Uruguaiana, São Borja, Itaqui Alegrete and Porto Alegre) in the years of 2007 and 2008. By testing hemagglutination inhibition (HI) were detected HI antibodies of Saint Louis and Ilhéus and other Flaviviruses included in the tests, as well as cross-reactivity for Flaviviruses. By HI test, 14.3% (254/1775) of animals were positive for Flavivirus, monospecific reactions were observed in 42.9% (109/254) serum samples, being that 78.9% (86/109) for St. Louis, 17.4% (19/109) for Ilhéus and 3.7% (4 / 109) for Rocio and cross-reactions were detected in 57.1% (145/254). Among the positives, there was no difference between the sexes. The age group ≥ 10 years old was the most affected with 35.4% (73/206). The animals used for the practice of sport were positive in 34.3% (87/254). The state of Paraná showed 16.3% (107/657) of reacting animals, followed by Rio Grande do Sul with 15.1% (142/939) and Santa Catarina with 2.8% (5/179). This study brings new data regarding the immunity of horses against Flaviviruses in Brazil, and confirms the wide distribution of St. Louis and Ilhéus and the diversity of Flavivirus in the country, as well as the apparent absence of clinical disease in horses infected with the virus studied. / As arboviroses são enfermidades que acometem o homem, equinos e outras espécies animais causando na sua maioria das vezes infecções que vão desde assintomáticas até neurológicas. Os Flavivirus são importantes arbovírus encontrados no Brasil. Foi realizado um estudo descritivo a partir de inquéritos sorológicos em 1775 equinos para detecção de anticorpos anti-Flavivirus no Estado do Paraná (Foz do Iguaçu, Maringá e Barracão), Santa Catarina (São Miguel do Oeste, Guaraciaba, Iraceminha, Dionísio Cerqueira, Guarujá do Sul e São Jose do Cedro) e Rio Grande do Sul (Uruguaiana, São Borja, Itaqui, Alegrete e Porto Alegre) nos anos de 2007 e 2008. Por meio de testes de inibição da hemaglutinação (IH) foram detectados anticorpos IH de Saint Louis e Ilhéus e outros Flavivirus incluídos nos testes, assim como reações cruzadas para Flavivirus. Pelo teste de IH, 14,3% (254/1775) dos animais foram reagentes para Flavivirus, reações monoespecíficas foram observadas em 42,9% (109/254) amostras de soro, sendo que 78,9% (86/109) para Saint Louis, 17,4% (19/109) para Ilhéus e 3,7% (4/109) para Rocio e reações cruzadas foram detectadas em 57,1% (145/254). Dentre os animais reagentes, não foi observada diferença entre os sexos. A faixa etária ≥ 10 anos foi a mais acometida com 35,4% (73/206). Os animais utilizados para a prática do esporte foram reagentes em 34,3% (87/254). O Paraná apresentou 16,3% (107/657) dos animais reagentes, seguido do Rio Grande do Sul com 15,1% (142/939) e Santa Catarina com 2,8% (5/179). Este estudo traz novos dados a respeito da imunidade de equinos contra Flavivirus no Brasil, e confirma a ampla distribuição de Saint Louis e Ilhéus e a diversidade de Flavivirus no País, bem como a aparente ausência de doenças em equinos infectados pelos vírus estudados.

La génétique inverse est devenue une méthode clé pour la production de virus à ARN génétiquement modifiés et pour comprendre les propriétés cellulaires et biologiques des virus. Cependant les méthodes les plus fréquemment utilisées, basées sur le clonage de génomes viraux complets dans des plasmides, sont laborieuses et imprévisibles. La première partie de cette thèse présente des études sur la mise au point d'un nouveau système de génétique inverse, appelé méthode ISA (Amplicons-Sous génomique-Infectieux), qui permet la génération, en quelques jours, de virus infectieux sauvages et génétiquement modifiés appartenant à trois familles différentes de virus à ARN simple brin de polarité positive, avec une grande maîtrise des séquences virales. Dans la deuxième partie de cette thèse, nous avons appliqué pour la première fois à un arbovirus (CHIKV), le ré-encodage des codons - une méthode développée récemment et très excitante pour le développement de vaccins vivants atténués. En utilisant une approche aléatoire de ré-encodage des codons qui attribue au hasard des codons sur la base de la séquence en acides aminés correspondante, nous avons mis en évidence des pertes importantes de fitness réplicatif sur des cellules de primates et d'arthropodes. La diminution du fitness réplicatif est en corrélation avec le degré de ré-encodage, une observation qui peut aider à la modulation de l'atténuation virale. En utilisant l'expérience acquise avec le CHIKV, nous avons transposé avec succès ce mécanisme d'atténuation au JEV et amélioré notre maîtrise du processus d'atténuation en utilisant une combinaison de la synthèse de novo et de la méthode ISA. / Reverse genetics has become a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but the most commonly used methods, based on the preparation of plasmid-based complete viral genomes, are laborious and unpredictable. The first part of this thesis presents studies relating to the development of a new reverse genetics system, designated the ISA (Infectious-Subgenomic-Amplicons) method, which enabled the generation of both wild-type and genetically modified infectious viruses belonging to three different families of positive, single stranded RNA viruses within days with great control of the viral sequences. In the second part of this thesis, we applied for the first time to an arbovirus (CHIKV), codon re-encoding - a recently developed and very exciting method for the development of live attenuated vaccines. Using a random codon re-encoding approach which randomly attributed nucleotide codons based on their corresponding amino acid sequence, we identified major fitness losses of CHIKV in both primate and arthropod cells. The decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. Detailed analysis of these observed replicative fitness losses indicated that they are the consequence of several independent re-encoding induced events. Using the experience acquired on the CHIKV, we successfully transposed this attenuation mechanism to JEV and improved our control of the attenuation process by using a combination of de novo synthesis and the ISA method.

Rondônia apresenta área com rica diversidade de artrópodes, porém pouco se conhece sobre a transmissão de arbovírus por estas espécies. O presente trabalho visou detectar arbovírus, por meio da RT-PCR e da Duplex RT-PCR, nas espécies de dipteros coletados no Estado, bem como caracterizá-los pela reação de sequenciamento. A RT-PCR e a Duplex RT-PCR detectaram as suspensões dos vírus Mayaro e Oropouche até 104 e 101 TCID50/mL, respectivamente, porém o vírus Dengue 2 em pools contendo menos de três mosquitos infectados foi negativa. O controle endógeno foi detectado em 66,8 % das amostras, sendo que, em pools contendo entre um e três mosquitos, a detecção foi aproximadamente metade da detecção nos pools contendo entre 11 e 15. Em 0,66 % dos pools foi encontrado o vírus Oropouche e em outros 0,66 %, o vírus Cacipacoré. O vírus Oropouche foi detectado em Coquillettidia sp. e Deinocerites sp., enquanto o vírus Cacipacoré foi encontrado em Anopheles sp. e Culex sp. As técnicas possibilitaram a detecção dos arbovírus pesquisados nos pools coletados em Rondônia. / The Rondônia state has an area with rich arthropods diversity although the knowledge about the arboviruses transmition for these species is poor. The present work aimed to detect arboviruses through RT-PRC and RT-PCR Duplex in the diptera species collected in the region as well as their characterization through the sequence reaction. The RT-PRC and RT-PCR Duplex detected the Mayaro and Oropouche virus suspensions until 104 e 101 TCID50/mL respectively, although it was negative for the Dengue 2 virus in pools containing less than three infected mosquitoes. The endogenous control was detected in 66,8 % of samples and from pools containing from one to three mosquitoes the detection rate was approximately half from that obtained from pools with 11 to 15 mosquitoes. Oropouche virus was found in 0,66 % of pools and Cacipacore virus also in 0,66 % of pools. Oropouche virus was detected in Coquillettidia sp. and Deinocerites sp. while Cacipacoré virus was found in Anopheles sp. and Culex sp. The techniques allowed the detection of examined arboviruses in the pools collected from Rondonia.

O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas. / Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.

Metropolitan mosquitoes: Understanding urban livestock keeping and vector-borne disease in growing tropical sites – The potential of sustainable control methods and the risks for emergence to Sweden.

Cellular T-cell intracellular antigen-1 related protein (TIAR) binds to the 3' terminal stem-loop of the West Nile virus minus-strand RNA [WNV 3'(-) SL RNA]. TIAR binding sites were previously mapped on loop 1 (L1) and loop 2 (L2) of the 3' (-) SL RNA and mutations of these sites in a WNV infectious clone inhibited virus replication. In the present study, data from in vitro binding assays suggested that multiple TIAR proteins bind to each WNV 3′ (-) SL RNA in a positively cooperative manner. The tertiary structure of WNV 3′ (-) SL RNA was predicted and it was suggested that L2 forms an exposed loop while L1 forms an embedded loop. We propose that TIAR binds first to L2 and that this interaction facilitates the binding of a second TIAR molecule to L1. Data from in vitro assays also showed that TIAR binds specifically to the WNV 3' (-) SL RNA but not to the complementary WNV 5' (+) SL RNA and that the C-terminal prion domain of TIAR contributes to RNA binding specificity. Immunoprecipitation experiments indicated that TIAR interacts with the WNV 3' (-) SL RNA in cells. Colocalization of TIAR and viral dsRNA in the perinuclear region of WNV-infected cells was visualized using a proximity ligation assay. In WNV-infected, TIAR-overexpressing cells, increased extracellular virus yields, intracellular viral protein and RNA levels, and an increased ratio of viral plus-strand RNA to minus-strand RNA were observed. These data suggest that TIAR enhances WNV plus-strand RNA synthesis from the minus-strand template. WNV infections induce small TIAR foci formation in primate cells but not rodent cells. The TIAR foci are located in the perinuclear region and differ in size and location from arsenite-induced stress granules (SGs). However, the small TIAR foci contain many SG components, such as G3BP, PABP, and eIF3A, but not HuR. Arsenite-induced SG formation is still inhibited by WNV infection in these cells. eIF2a phosphorylation was observed in some infected cells that contained WNV-induced TIAR foci but viral NS3 protein accumulation was not inhibited. The data suggest that WNV-induced TIAR foci in primate cells are not canonical SGs.

O vírus da Zika (ZIKV) é um arbovírus emergente da família Flaviviridae, do gênero Flavivirus transmitido por mosquitos Aedes. Apesar da sua importância emergente na saúde pública, ainda pouco se conhece sobre os mecanismos moleculares envolvidos no ciclo replicativo do ZIKV em célula humanas. Assim, o objetivo geral deste estudo foi caracterizar a distribuição subcelular do ZIKV na célula hospedeira e elucidar fatores celulares que regulam o tráfego intracelular de proteínas envolvidos nesses processos. Mais especificamente, determinar os compartimentos celulares que servem de plataforma de montagem para o ZIKV. Além disso, também verificar se o funcionamento da maquinaria Endosomal Sorting Complexes Required for Transport (ESCRT) é requerido no ciclo replicativo de ZIKV. Para identificar a localização subcelular do ZIKV, foram utilizados diferentes marcadores celulares, e, de acordo com os resultados, foi demonstrado que com 3 horas pós infecção (h. p. i.) ocorre colocalização de proteínas do ZIKV com um marcador de endossomo primário, enquanto que com 15h p.i. já é possível detectar proteínas virais no Retículo Endoplasmático (RE). Subsequentemente, com 27h p.i. o ZIKV direciona-se para o complexo de Golgi. Juntos, esses resultados indicam o direcionamento do ZIKV através da via secretória ao longo do tempo. Além disso, foi testado o envolvimento da maquinaria dos ESCRTs por meio do silenciamento da expressão da proteína TSG101 de ESCRT-I em células infectadas com ZIKV. Os resultados obtidos, sugerem que ESCRT-I tem participação importante na replicação do ZIKV, ocorrendo a diminuição dos títulos virais quando TSG101 é depletada da célula. Em conjunto, os resultados permitem concluir que ao longo da infecção o ZIKV encontrase associado aos compartimentos da via secretória inicial (RE e complexo de Golgi), e que a proteína TSG101 de ESCRT-I exerce papel importante na replicação viral. Sendo assim, esse estudo possibilitou um melhor entendimento sobre a dinâmica de replicação do ZIKV em células humanas. / Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, of the genus Flavivirus that is transmitted by Aedes mosquitoes. Despite its emerging importance in public health, little is known about the molecular mechanisms involved in the replicative cycle of ZIKV in human cells. Thus, the general objective of this study was to characterize the subcellular distribution of the ZIKV in the host cell and to elucidate cellular factors that regulate the intracellular trafficking of proteins involved in these processes. More specifically, to determine the cellular compartments that serve as assembly platforms for the ZIKV. In addition, the study aimed to verify if the functioning of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is required in the replicative cycle of ZIKV. In order to identify the subcellular localization of ZIKV, different intracellular markers were used, and, according to the results, it was demonstrated that at 3 hours post infection (h. p. i.) ZIKV proteins colocalize with an early endosome marker, whereas within 15h p.i. it is already possible to detect newlysynthesized viral proteins in the endoplasmic reticulum (ER). Subsequently, within 27h p.i., the ZIKV is directed to the Golgi complex. Together, these results delineate the targeting of ZIKV proteins through the secretory pathway over time. In addition, the involvement of the ESCRT machinery was tested by knocking down the expression of ESCRT-I protein TSG101 in ZIKV-infected cells. The results obtained suggest that ESCRT-I plays an important role in ZIKV replication, with viral titers decreasing when TSG101 levels are depleted in the cell. Together, the results allow us to conclude that ZIKV is associated with the initial secretory pathways (RE and Golgi complex) throughout the infection, and that the ESCRT-I TSG101 protein plays an important role in viral replication. Thus, this study contributes to a better understanding of the dynamics of ZIKV replication in human cells.

abstract: Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV. / Dissertation/Thesis / Masters Thesis Biology 2015

abstract: Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016