[Truncate abstract] Flaviviruses are small, positive-stranded RNA viruses belonging to the family Flaviviridae. Flavivirus infection in humans could cause diseases ranging from febrile illnesses to fatal encephalitis. Mice provide a useful small animal model to study flavivirus-induced encephalitis in humans since mice also develop encephalitis during flavivirus infection. Some strains of mice have been shown to be resistant to flavivirus challenge and this resistance is conferred by a single autosomal dominant gene, designated as Flvr. Recently, OAS1b gene has been identified to be a gene candidate for Flvr. Several congenic resistant mouse strains have been developed by introducing resistance genes from outbred or wild mice onto the genetic background of susceptible C3H mice. These new resistant strains that carry different allelic variants at the Flv locus include C3H/PRI-Flvr (RV), C3H.MOLD-Flvmr (MOLD) and C3H.M.domesticus-Flvr-like (DUB), the latter two being developed in the same laboratory in which the work described in this thesis was accomplished. Preliminary studies in this laboratory found that flavivirus resistant mice are vulnerable to certain flavivirus infections, particularly when challenged by intracerebral (i.c.) route. Intracerebral (i.c.) challenge with flaviviruses such as West Nile virus (WNV) Sarafend strain and Kunjin virus (KUNV) MRM16 strain were found to induce high mortality in flavivirus resistant mice while infection with Murray Valley encephalitis virus (MVEV) OR2 strain did not cause any apparent disease in the same mice. ... Thus, it can be concluded that CD8+ T cells exerted harmful effect to resistant DUB mice during KUNV i.c. infection by producing excessive IFN[gamma] that could be toxic, causing functional loss of the CNS cells. It was shown from in vitro studies that WNV had the highest tropism for macrophages and dendritic cells, followed by KUNV. MVEV however did not replicate well in these cells. This combined with the data from the in vivo studies indicates that macrophages might be involved in the pathogenesis of intraperitoneal (i.p.) infection of WNV but not KUNV and MVEV. The reason for this could be that the production of KUNV in macrophages may not be high enough to induce viraemia and subsequent fatal encephalitis in mice. In contrast, MVEV appears to use different mechanism or cells for virus dissemination. Although macrophages may not be involved in KUNV pathogenesis after i.p. infection, the fact that macrophages support KUNV replication in vitro may indicate the possibility that blood-borne macrophages were recruited to the brain where they can get infected with KUNV during i.c. infection and therefore could participate in KUNV pathogenesis in DUB mice. This study provides evidence for the first time on the detrimental effect of host antiviral immunity and inflammatory mediators during flavivirus i.c. infection in resistant mice. However, it also launches a new question on the selective cell tropism of KUNV versus MVEV responsible for inducing different pattern of immune responses and consequently leading to different outcomes of infection in resistant mice.

Entre els anys 2001 i 2005 es van capturar i analitzar 72.895 femelles de mosquits (Diptera: Culicidae) i 6.871 de flebòtoms (Diptera: Psychodidae) en les estacions d'abundància amb l'objectiu de detectar la presència de genoma d'arbovirus en diferents àrees d'Espanya, en especial en zones humides. L'estudi forma part d'un de més general que tracta de la transmissió d'arbovirus en quatre dels aiguamolls més importants d'Espanya que es troben a Girona, Barcelona, Tarragona i Huelva.Els insectes es van recollir amb esquer humà, amb trampes de CO2 i amb trampes CDC i es van agrupar en pools segons la data de captura, la localitat i l'espècie. Pel que fa als flebòtoms, les mostres es van obtenir a partir del 2002 a Barcelona i Huelva i es van identificar com a subfamília.Els culícids es van agrupar en 4.723 pools i pertanyien a 20 espècies dels gèneres Anopheles, Aedes, Ochlerotatus, Culex, Culiseta, Coquillettidia i Uranotaenia i els flebòtoms es van agrupar en 236 pools com a tals.Mentre es duia a terme l'estudi, es va detectar el mosquit invasor -Aedes (Stegomyia) albopictus (Skuse)- per primera vegada a Espanya, concretament a Sant Cugat del Vallès, durant l'estiu del 2004. Mitjançant inspeccions immediatament posteriors es va comprovar l'existència d'importants poblacions a la zona, i se'n va confirmar l'establiment. Aquesta és la primera notificació de l'espècie esmentada a la península Ibèrica. Es van analitzar totes les femelles capturades posteriorment a l'àrea d'estudi.L'espècie de culícid més abundant va ser Ochlerotatus caspius (40,9 %), seguida de Culex pipiens (32,3 %), Culex theileri (10,9 %), Anopheles atroparvus (6,6 %), i Culex modestus (4,6 %).Es van analitzar homogenats dels vectors per detectar directament ARN d'arbovirus dels gèneres Alphavirus, Flavivirus i Phlebovirus. No s'ha trobat ARN d'arbovirus patògens coneguts. En el cas dels mosquits, 111 pools van ser positius a Flavivirus, l'únic gènere detectat en aquest grup taxonòmic. Les seqüències de Flavivirus identificades són diferents de qualsevol Flavivirus de mosquit conegut i majoritàriament properes al virus Kamiti River (KRV) o al virus cell fusing agent (CFA), excepte en dos pools d'Andalusia que es troben properes al grup de virus transmesos per artròpodes. Per a totes les zones i espècies, es va calcular l'estimació del màxim de versemblança de la taxa d'infecció o the maximum likelihood estimation infection rate (MLE). Ae. albopictus tingué la MLE més alta, de 47,14, seguida per Aedes vexans amb 43,67 en el conjunt de l'àrea d'estudi. Per sota d'aquestes espècies hi havia Culiseta annulata, amb 36,00. Les espècies més abundants, Oc. caspius i Cx. pipiens, va obtenir valors MLE baixos (0,94 i 0,38 respectivament) en el conjunt de tota l'àrea.En el cas dels flebòtoms, 10 pools (9 dels quals de Barcelona) van donar positiu a Flavivirus semblants a Culex Flavivirus (CxFV). És la primera vegada que es troba genoma d'aquest gènere en flebòtoms de fora de l'Àfrica. En 8 pools de Barcelona es va trobar un Phlebovirus similar al complex Nàpols i al virus Massilia.Cal assenyalar que en el cas d'alguns mosquits, en especial en mostres dels gèneres Aedes i Ochlerotatus, el genoma detectat probablement eren seqüències d'ADN integrades en el genoma dels mosquits. Aquest fet l'han observat recentment altres autors.El 2006, seguint amb la campanya de detecció d'arbovirus, es va trobar, entre 436 pools dels aiguamolls de Catalunya i 9 espècies de mosquit, un de positiu a un Flavivirus identificat com a virus Usutu (USUV) en un pool de 3 Cx. pipiens obtingut a Viladecans (Barcelona). Les dades d'homologia van mostrar que la soca espanyola pertanyia a l'USUV però que era més propera a mostres africanes d'USUV que a les obtingudes a Europa central. / With the aim of assessing the presence of arbovirus genome in vectors in different areas, especially wetlands, in Spain, a total of 72,895 female mosquitoes (Diptera: Culicidae) and 6,871 sandflies (Diptera: Psychodidade) were trapped during their season of abundance, and analyzed between the years 2001 and 2005. The study formed part of general arbovirus transmission research in four of the most important wetlands in Spain in the provinces of Girona, Barcelona, Tarragona, and Huelva.Insects were collected using human bait, CO2 traps, or light traps, and they were pooled according to date of collection, location, and species. In the case of sandflies, the period of study started in 2002 in Barcelona and Huelva and species were not identified. Mosquitoes were sorted into 4,723 pools belonging to 20 Culicidae species from the Anopheles, Aedes, Ochlerotatus, Culex, Culiseta, Coquillettidia, and Uranotaenia genera. Sandflies were sorted into 236 pools as a whole.During the study, the invasive mosquito Aedes (Stegomyia) albopictus (Skuse) was detected for the first time in Spain, in Sant Cugat del Vallès during August 2004. Dense populations of adults and larvae were found in subsequent surveys, confirming the establishment of this species in this area. This is the first report of the establishment of Ae. albopictus in the Iberian Peninsula. All captured females in the studied area belonging to this species were analyzed.The most abundant species was Ochlerotatus caspius (40.9 %), followed by Culex pipiens (32.3 %), Culex theileri (10.9 %), Anopheles atroparvus (6.6 %), and Culex modestus (4.6 %).Arboviral RNA was directly detected from vector homogenates for the genera Alphavirus, Flavivirus, and Phlebovirus. No arboviral RNA from known pathogenic arboviruses was found. In the case of mosquitoes, 111 pools tested positive for unknown mosquito Flavivirus, the only genus detected in this taxonomic group. The Flavivirus sequences identified were different from all known Flavivirus mosquito viruses, but very close to Kamiti River virus (KRV) or cell fusing agent virus (CFA) with the exception of two pools from Andalusia, close to the group of arthropod borne viruses. The maximum likelihood estimation infection rate (MLE) was calculated for all regions and species. Ae. albopictus had the highest MLE at 47.14, followed by Aedes vexans with 43.67 over the entire area. These species were followed by Culiseta annulata, with 36.00. The most common species, Oc. caspius and Cx. pipiens, had low MLE values -0.94 and 0.38, respectively- over the area as a whole.In the case of sandflies, 10 pools (9 in Barcelona) tested positive for a Flavivirus similar to Culex Flavivirus (CxFV) being this, the first time that Flavivirus genome is detected in sand flies outside Africa. Phlebovirus viruses similar to Naples complex and Massilia were found in 8 sandflies pools, all from Barcelona area.In some samples of mosquitoes, especially in genera Aedes and Ochlerotatus, detected genome was probably DNA sequences integrated in the mosquito genome as has been observed recently by other authors.In 2006, surveillance monitoring samples carried out in Catalonia detected, in 436 pools belonging to 9 mosquito species, a positive for Flavivirus identified as Usutu virus (USUV) in a pool of 3 Cx. pipiens obtained from the town of Viladecans, Barcelona. The homology data showed that the Spanish strain belongs to USUV species and is more related to the African USUV isolates than to central European isolates.

Reproduction de : Thèse d'exercice : Pharmacie : Nantes : 2008. / Bibliogr.

Made available in DSpace on 2014-09-09T12:30:35Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 41.pdf: 8046601 bytes, checksum: e06703afffad9699a86a2084978dbe84 (MD5) Previous issue date: 2012 / A meningite asséptica é uma síndrome infecto-contagiosa, estabelecida após inflamação das meninges. A etiologia mais comum desta síndrome é a viral, e os enterovírus são responsáveis por mais de 80% dos casos em que o agente etiológico é identificado. Outros agentes etiológicos virais envolvidos são Arbovírus (West Nile virus, Vírus da Encefalite Japonesa e Vírus da Encefalite de Saint Louis), vírus da caxumba, herpersvírus e adenovírus, entre outros. A primeira etapa deste estudo teve como objetivo o aprimoramento do diagnóstico dos casos de meningite asséptica e meningoencefalite com a tentativa de detecção molecular de enterovírus em 267 amostras de LCR recebidas entre 2008 e 2009, negativas para o isolamento viral em culturas de células. A extração direta do LCR seguida de síntese de cDNA e PCR foi capaz de detectar enterovírus em 59 amostras de LCR (22,1%). Estes resultados foram confirmados por sequenciamento nucleotídico parcial e demonstram que a pesquisa do genoma viral diretamente dos LCRs é apropriada para um aumento na possibilidade de detecção de enterovírus neste tipo de amostra clínica. O objetivo da segunda etapa deste estudo foi analisar o potencial papel etiológico dos flavivírus em casos de meningite asséptica e meningoencefalite. As 208 amostras negativas na etapa anterior foram utilizadas nesta etapa. Para tanto, foram desenvolvidas duas abordagens moleculares para detecção de flavivírus: semi-Nested-PCR e PCR convencional. Iniciadores foram desenhados para as duas técnicas. / A meningite asséptica é uma síndrome infecto-contagiosa, estabelecida após inflamação das meninges. A etiologia mais comum desta síndrome é a viral, e os enterovírus são responsáveis por mais de 80% dos casos em que o agente etiológico é identificado. Outros agentes etiológicos virais envolvidos são Arbovírus (West Nile virus, Vírus da Encefalite Japonesa e Vírus da Encefalite de Saint Louis), vírus da caxumba, herpersvírus e adenovírus, entre outros. A primeira etapa deste estudo teve como objetivo o aprimoramento do diagnóstico dos casos de meningite asséptica e meningoencefalite com a tentativa de detecção molecular de enterovírus em 267 amostras de LCR recebidas entre 2008 e 2009, negativas para o isolamento viral em culturas de células. A extração direta do LCR seguida de síntese de cDNA e PCR foi capaz de detectar enterovírus em 59 amostras de LCR (22,1%). Estes resultados foram confirmados por sequenciamento nucleotídico parcial e demonstram que a pesquisa do genoma viral diretamente dos LCRs é apropriada para um aumento na possibilidade de detecção de enterovírus neste tipo de amostra clínica. O objetivo da segunda etapa deste estudo foi analisar o potencial papel etiológico dos flavivírus em casos de meningite asséptica e meningoencefalite. As 208 amostras negativas na etapa anterior foram utilizadas nesta etapa. Para tanto, foram desenvolvidas duas abordagens moleculares para detecção de flavivírus: semi-Nested-PCR e PCR convencional. Iniciadores foram desenhados para as duas técnicas. O uso desses iniciadores possibilitou a amplificação do genoma de flavivírus utilizados como controle, mostrando serem ferramentas úteis para a detecção desses vírus. Apesar disso, nenhuma amostra de LCR foi positiva para flavivírus. Diante destes resultados, da possibilidade da circulação silenciosa de WNV ou de sua entrada iminente no país, é evidenciada a necessidade de estudos adicionais sobre este vírus e outros flavivírus nestes casos. Na terceira etapa deste estudo, foi realizada a análise da variabilidade genética de echovírus 30 envolvidos em surtos e casos esporádicos de meningite asséptica entre 1998 e 2008, através da análise filogenética do gene completo (876 nt) da VP1 de 48 E30 isolados. Além da comparação das amostras entre si, foram feitas comparações destas com a VP1 da cepa protótipo Bastianni e também com outras cepas isoladas em diversos países. Durante o período do estudo, E30 foi o principal enterovírus envolvido nos casos de meningite asséptica e meningoencefalite no Brasil (58,6% dos 302 enterovírus isolados), tendo sido o agente etiológico de seis surtos. As sequências de VP1 de E30 segregaram em três Grupos distintos e sete subgrupos, cujos agrupamentos estavam fortemente associados ao ano de isolamento. A divergência de sequências nucleotídicas entre os E30 isolados variou de 0,2-13,8%. Não foi definido um ancestral comum direto para este conjunto de isolados de E30. O isolado 39-SC- BA-07 estava geneticamente relacionado com cepas de E30 isoladas no Pará (3,2-4,6% de divergência) e pode ser originário de uma destas cepas. Os isolados do Grupo I estavam geneticamente relacionados com uma sequência de E30 isolada em 1997 nos Estados Unidos (2,4-5,7%), sendo provável que eles possuam um ancestral comum. Dois E30 isolados na Argentina em 2007 mostraram estreita relação com os isolados do Grupo III (5,8-6,0%), podendo ter sido originados a partir de um dos isolados deste Grupo III.

The last five years have shown us the profound impact that SARS-CoV-2 pandemic has had on human kind and made us aware of the dangers that emerging pathogens can present. The goal of this dissertation is to use mathematical models in connection with data to uncover mechanistic interactions governing viral infections. To acquire a holistic understanding of the impact of viral infections, it is necessary to develop mathematical techniques and models that bridge knowledge on multiple biological scales. This dissertation explores the relationship between within-host virus dynamics, the environment and the between-host viral transmission. We will validate the models against data from SARS-CoV-2 infections, and data from infections with an emerging pathogen, the Usutu virus. Our models of SARS-CoV-2 infection looked at the relationship between infectious virus and viral RNA in the body and in the environment. Using golden hamster data and within-host mathematical models, we determined that infectious virus shedding early in infection correlates with transmission events, shedding of infectious virus diminishes late in the infection, and high viral RNA levels late in the infection are a poor indicator of transmission. We further showed that viral infectiousness increases in a density dependent manner with viral RNA and that their relative ratio is time-dependent. Such information is useful for designing interventions. Our models of Usutu virus infection looked at differences between different virus strains during bird infections. Within-host models applied to data showed heterogeneity in viral strain dynamics, and correlated high basic reproductive number with short infected cell lifespan (indicative of immune responses) and correlated low basic reproductive number with low viral peaks and longer lasting viremia (due to lower infection rates and high infected cell lifespan). We expanded the models to investigate multiscale dynamics connecting within-host scale, bird-to-vector transmission scale, and vector-borne epidemiological scale. One important direction of this dissertation is the investigation of uncertainty in parameter estimation and overall model identifiability. We conducted identifiability studies (using several theoretical tools) in the multiscale models of Usutu virus infection and in several within-host influenza models. Model identifiability is critical to the reproducibility of modeling results in any biological systems. In this dissertation, we will show how insights from such analyses inform both modeling practices and experimental design. / Doctor of Philosophy / The last five years have shown us the profound impact that SARS-CoV-2 pandemic has had on human kind and made us aware of the dangers that emerging pathogens can present. Within-host mathematical models are tools that can be used to study the dynamics of virus infections. These models help us gain an understanding of biological quantities of interest, relationships between biological processes in a quantitative and qualitative ways, and disease outcome. However, to acquire a holistic understanding of the impact of viral infections, it is necessary to develop mathematical tools and models that bridge knowledge on multiple biological scales. This dissertation explores the relationship between virus infection characteristics over time in a single host and larger biological scales including virus' release into the environment and spread of virus between hosts. Biological and public health insights about SARS-CoV-2 and Usutu virus were gained through these modeling efforts.

Kyasanur Forest disease virus (KFDV) of the Flaviviridae virus family has caused seasonal infections and periodic outbreaks in Karnataka, India. First identified in 1957, KFDV annually infects 400-500 people and has a fatality rate of 3-5%; there are no approved antivirals and the existing licensed vaccine’s effectiveness appears to be questionable. Many tools for KFDV research are limited and this work sought to develop methods for analysing antivirals, including interferon (IFN)-α/β species. The BHK-21 (ATCC) cell line allowed for high virus propagation and distinguishable cytopathic effects (CPE) for determining antiviral effectiveness. The additional tool of a reverse genetics system expressing a full-length cDNA KFDV genome with a GFP reporter failed to propagate, despite numerous GFP genome-insertion strategies. The clinically approved IFN-α2a or IFN-α2b has had variable success at combatting flavivirus diseases in people, especially in the immuno-compromised. The continued passaging of KFDV-infected cells with repeated IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production. IFN-αspecies, αWA and α were more effective than IFN-α2a and α2b at reducing KFDV; however dose ranges indicated that while low concentrations could limit CPE, higher concentrations were needed to inhibit virion release. Avoidance of IFN-α/β through Jak/STAT signalling repression was attributed to the NS5 protein, specifically the RdRp domain based on data obtained with luciferase and vesicular stomatitis virus (VSV) recovery assays. However, the mechanism appears to act subsequently to STAT1/2 activation without NS5 binding to any Jak/STAT components. A non-infectious, replicative system serving as a platform for antiviral drug testing against KFDV in a high throughput manner could only provide luciferase signals when the NS proteins capable of driving replication, were supplied in cis (subgenomic) but not in trans (antigenome). To conclude, IFN-α species such as IFN-αWA may be better suited than the licensed IFN-α2a for treatment of KFDV infections; however, IFN effects appear to be subdued in vitro due to the actions of the NS5 protein. While IFN may not be a successful antiviral against KFDV, the work in this thesis provides a foundation for evaluating other potential anti-KFDV therapeutics. / February 2016

Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré. / Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.

INTRODUÇÃO: Em grandes cidades, como São Paulo, a pequena porcentagem de matas existentes é representada pelos parques municipais. Estas áreas, além de representarem ambientes de lazer para as pessoas, albergam uma enorme diversidade biológica, desde mosquitos até aves e mamíferos. A interação destas espécies favorece a circulação de Flavivirus, causadores de importantes doenças humanas, que têm nas aves um importante reservatório. Atribui-se às aves migratórias o papel de carreadoras dos vírus, já que as mesmas percorrem longas distâncias para completar seu ciclo biológico. A chegada de aves do Hemisfério Norte ocorre em alguns Parques, o que favoreceria a dispersão de alguns vírus, como o Vírus do Nilo Ocidental, já que nestas áreas estão presentes mosquitos potencialmente vetores. OBJETIVOS: identificar infecção por Flavivírus nas aves dos parques municipais de São Paulo. MÉTODOS: De Março de 2012 a Janeiro de 2013, foram coletadas amostras de swab de cloaca, orofaringe e sangue de aves capturadas em redes de neblina de duas áreas do município de São Paulo: Parque Anhanguera e Fazenda Castanheiras/APA Bororé Colônia. As aves foram anilhadas e liberadas após a coleta do material. Foi realizada técnica de RT-PCR em tempo real, utilizando iniciadores genéricos que amplificam fragmento do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento. RESULTADOS: Foi capturado um total de 231 aves, em sua maioria da Ordem Passeriformes. De um total de 463 amostras, nenhuma amostra apresentou presença de RNA viral. DISCUSSÃO: Em se tratando de alguns Flavivírus, os passeriformes são considerados os reservatórios mais competentes no ciclo de transmissão, pois atingem altos níveis de viremia. Cabe ressaltar que algumas espécies desta ordem, de ocorrência na cidade de São Paulo, já foram identificadas como portadoras dos vírus Rocio e Ilhéus. A ausência de positividade é esperada, pois embora altamente sensível, a técnica de PCR depende do estado de viremia das aves, que é curta. CONCLUSÃO: Os parques municipais são áreas que aproximam aves, mosquitos e humanos, pelo papel ambiental e de lazer que os mesmos representam. Este fato classifica estas áreas como locais com potencial de transmissão de Flavivirus, o que torna importante a continuação este estudo, aumentando as áreas de abrangência, para conhecer os Flavivírus circulantes e realizar vigilância para vírus que podem ocasionar problemas de Saúde Pública / INTRODUCTION: In big cities, as São Paulo, the little percentage of existing forests is represented by the municipal parks. These áreas, besides acting as entertainment environments to the users, promote a huge biodiversity, including mosquitoes, birds and mammals. These species interaction promotes Flavivirus circulation, viruses responsible for important human diseases. The avian species are important reservoirs for these viruses, specially the migrating birds that can fly for long distances, carrying these viruses to several areas. In some parks, the arrival of migrating birds from the North Hemisphere is documented, fact that can support some viruses dispersion, for example, the West Nile Vírus, considering that in these areas potencial vector for this virus can be found. OBJECTIVES: detect Flavivirus infection in birds captured in municipal parks of São Paulo city. METHODS: from March, 2012 to January, 2013, oropharyngeal and cloacal swabs, and blood samples were collected from birds captured in mist-net webs located in two municipal areas: Anhanguera Park and Castanheiras Farm/APA Bororé Colônia. The birds were ringed and released after samples collection. The real time RT-PCR was performed, using generic primers which amplify NS5 gene fragment. Positive samples were forwarded to sequence analysis. RESULTS: a total of 231 birds were capture, in which the majority belongs to Passeriforms order. Of 463 samples collected, all samples were negative for the viral RNA presence. DISCUSSION: when talking about Flaviviruses, the passeriforms are considered the most competent reservoirs in the transmission cycle, because they can achieve great viremia levels. Some passeriforms species, endemic in São Paulo city, were identified as Rocio and Ilhéus viruses carriers in previous studies. The negativity is expected, once the Real Time RT-PCR, although highly sensitive, depends on the viremia duration, which is short in avian species. CONCLUSION: the public parks are areas which encloses birds, mosquitoes and the human being, for the environmental and entertainment role they play. This fact classifies these parks as areas with great potencial of Flavivirus transmission, ressalting the great importance to continue this study, increasing the number of areas, to detect the circulating Flavivirus and to perform surveillance for viruses which can cause public health problems

Le virus Zika (ZIKV) est un phénomène épidémiologique sans précédent qui surprit le monde entier. Pendant de nombreuses années, il fut considéré comme un virus anodin responsable d’une poignée d’infections humaines, auto-limitées et bénignes, en Afrique et en Asie du Sud-est. Mais, après des décennies de propagation silencieuse, une première épidémie éclata en Micronésie en 2007 - tel un signal d'alarme. Quelques années plus tard, une soudaine épidémie de ZIKV de plus grande ampleur se déclara dans les îles du Pacifique avant d'atteindre le Brésil en 2015. Au cours de cette période, Zika fut associé à de graves complications neurologiques, mettant en évidence son fort potentiel pathogène pour l'homme. Depuis son émergence, plus de 80 pays et territoires ont été touchés par la pandémie de ZIKV, désormais reconnu comme un virus neurotrope et tératogène. L'association des souches contemporaines de ZIKV à des formes graves de maladie chez l'homme, qui n'ont jamais été signalées auparavant, a soulevé l'hypothèse d'une pathogénicité nouvellement acquise. Ainsi, mes travaux de doctorat visaient à déterminer si l'ampleur de l'épidémie actuelle pouvait en partie avoir été facilitée par des facteurs viraux qui auraient renforcé la fitness du ZIKV. À cette fin, mon projet de recherche s'est concentré sur l'identification des facteurs moléculaires viraux impliqués dans la pathogénicité du virus Zika chez l’homme à partir du développement de clones moléculaires. / Zika virus (ZIKV) is an unprecedented epidemiological phenomenon which surprised the world. For many years, it was considered a trivial virus responsible for only a handful of human infections, self-limited and benign, in Africa and Southeast Asia. But then, after decades of silent spread, a first epidemic broke out in Micronesia in 2007 – like a warning signal. A few years later, a sudden Zika outbreak of larger scale occurred in the Pacific islands before reaching Brazil in 2015. During this period, Zika was associated with severe neurological complications, highlighting its serious pathogenic potential for humans. Since its emergence, more than 80 countries and territories have been affected by the ZIKV pandemic, which is now recognized as a neurotropic and teratogenic virus. The association of contemporary ZIKV strains with severe forms of disease in humans, that have never been reported before, has raised the hypothesis of newly acquired pathogenicity. In this regard, my doctoral research aimed to determine whether the scope of the current epidemic was partly facilitated by viral factors that improved ZIKV fitness. To this end, my research project focused on the identification of the viral molecular factors involved in Zika virus pathogenicity in humans based on the development of molecular clones.

Introdução: Dos arbovírus transmitidos no Brasil, os Flavivirus apresentam destaque por causarem o maior número de infecções e doenças no homem e pela gravidade das doenças que provocam. A cidade de São Paulo possui áreas verdes, representadas por parques municipais em que existem lagos, aves e mamíferos. Esses locais são frequentados pela população e podem servir de refúgio para mosquitos que infestam a área urbana. Alguns trabalhos revelam a ocorrência de espécies de mosquitos conhecidas como vetoras de Flavivirus em tal cidade, o que pode favorecer o aparecimento de casos e transmissão de doenças causadas por este tipo de vírus. No entanto, a presença de Flavivirus infectando tais artrópodes não é conhecida. Objetivo: Identificar presença de Flavivirus em mosquitos (Diptera: Culicidae) provenientes de áreas verdes da cidade de São Paulo. Método: Sete parques municipais localizados em diferentes regiões da cidade foram selecionados para o estudo. Foram realizadas coletas de mosquitos mensais em cada parque no período de março de 2011 e fevereiro de 2012. As armadilhas utilizadas foram: aspirador, Shannon e CDC-CO2. Os mosquitos foram transportados para o laboratório em gelo seco, identificados em mesa fria e agrupados em \"pools\" de até 10 fêmeas não ingurgitadas, segundo espécie, data e local de coleta. Fêmeas ingurgitadas foram armazenadas individualmente. As amostras foram submetidas à extração de ácidos nucleicos, seguida de RT-PCR em tempo real para amplificação de fragmento de aproximadamente 200pb do gene NS5 de Flavivirus. Amostras positivas foram encaminhadas para sequenciamento e análises filogenéticas. Resultados: Dentre as 5.213 fêmeas não ingurgitadas (818 pools) e as 778 fêmeas ingurgitadas coletadas, ocorreu presença de onze gêneros de Culicídeos, sendo mais frequentes Culex e Aedes. Dezenove pools de fêmeas não ingurgitadas obtiveram resultado positivo para Flavivirus. Análises filogenéticas mostraram presença de RNA relacionado à Aedes flavivirus (AEFV) em dois pools de Aedes e à Culex flavivirus (CxFV) nos demais pools, todos de Culex. Conclusões: CxFV e AEFV estão presentes em mosquitos dos gêneros Culex e Aedes, respectivamente, coletados em parques municipais da cidade de São Paulo. Embora flavivírus patogênicos ao homem não tenham sido encontrados no presente trabalho, recomenda-se vigilância de flavivírus nas áreas estudas, visto que elas possuem presença de mosquitos vetores e hospedeiros vertebrados, ou seja, elementos necessários para a transmissão destes vírus. / Introduction: Among the arboviruses transmitted in Brazil, Flavivirus are noteworthy once they cause the largest number of infections and diseases in humans and also because they can cause serious illnesses. In the city of São Paulo there are green spaces, represented by city parks in which lakes, birds and mammals are found. The population visits those places, which can also be a refuge for mosquitoes that infect the urban area. Some studies reveal the occurrence of mosquitoes species known to be vectors of Flavivirus in this city, what can contribute to the emergence of cases and transmission of diseases caused by this kind of virus. However, the presence of Flavivirus infecting those arthropods is not known. Objective: To Identify infection by Flavivirus in mosquitoes (Diptera: Culicidae) collected in São Paulo\'s city parks. Method: Seven city parks located in different places of the city were selected for the study. Monthly mosquito collections were carried out in each park from March 2011 to February 2012. The traps employed were aspirator, Shannon and CDC-CO2. The mosquitoes were transported to the laboratory on dry ice. Identification was performed in a chill table. Up to 10 non-engorged females were pooled according to specie, date and place of collection. Engorged females were stored individually. Nucleic acids were extracted and submitted to real time RT-PCR that amplifies a 200pb fragment of NS5 gene of Flavivirus. Positive samples were sequenced and phylogenetic analyses were performed. Results: Eleven genus of Culicidae were found among the 5,213 non engorged females (818 pools) and the 778 engorged females. Culex and Aedes were the most frequent collected genus. Nineteen non engorged female pools had positive results for the presence of Flavivirus RNA. Phylogenetic analyses showed the RNA found was related to Aedes flavivirus (AEFV) in two pools of Aedes and to Culex flavivirus (CxFV) in the other pools, all of them consisting of Culex. Conclusion: CxFV and AEFV are present in mosquitoes Culex and Aedes, respectively, collected in São Paulo\'s city parks. Flavivirus of medical importance were not detected. Although, once there are species of mosquitoes that can act as vectors of pathogenic Flavivirus and vertebrate hosts, which are required for the transmission cycle of those virus, surveillance is recommended in the studied areas.