Phleboviruses are emerging zoonotic pathogens which constitute a global threat to human and animal health. The mosquito-borne Rift Valley fever virus (RVFV) is a widespread problem across the African continent and causes regular deadly outbreaks in ruminants. The recently emerged severe fever with thrombocytopenia syndrome virus (SFTSV) is a serious human public health concern in China which has rapidly spread to Japan and Korea with fatality rates as high as 16-30%. Phleboviral cell entry is mediated by two viral glycoproteins: the class II fusion protein Gc and the lesser known Gn. Initial cell attachment is glycan dependent and the penetration into the cell cytoplasm is mediated by the Gc fusion protein which catalyses viral and cell membrane merger. The entry mechanism is not well understood from a structural perspective which limits mechanistic insights. The purpose of this thesis is to further our understanding of the cell entry process by filling in the missing structural information on the phleboviral glycoprotein layer. To this end, an integrated structural approach using cryo-EM and X-ray crystallography was adopted. The crystal structure of the Gn ectodomain is presented which reveals an unprecedented structural relationship with seemingly unrelated viruses. Single-particle cryo-EM and localized reconstruction reveal the glycoprotein layer of the RVFV and a pseudo-atomic model of the RVFV is presented. The assembly shows the shielding of the Gc fusion protein and suggests that the Gn functions as a fusion chaperone. The post-fusion crystal structure of the Gc protein from SFTSV further consolidates a mechanism of membrane fusion by class II fusion proteins. Finally, preliminary data on receptor binding and mechanism of antibody mediated neutralization are presented. The work presented herein provides a novel platform for studying and understanding entry and assembly of phleboviruses as well as the design of novel therapeutics.

Les phlébotomes sont les vecteurs reconnus de plusieurs arbovirus, en particulier du genre Phlebovirus, ainsi que de parasites du genre Leishmania. Les infections par les phlébovirus sont responsables chez l’homme de maladies décrites depuis longtemps, pourtant ils demeurent méconnus, avec en particulier un manque de données épidémiologiques et d’outils de diagnostic.Dans une première partie, des études de séroprévalence nous ont permis d’aborder l’impact en santé publique, dans le sud-est de la France, de deux phlébovirus connus pour leur pouvoir pathogène chez l’homme, Toscana virus (TOSV) et Sandfly fever Sicilian virus (SFSV). Pour ce dernier, des anticorps spécifiques (IgG) ont été détectés dans moins de 1% des sérums testés, ce qui suggère que SFSV joue un rôle mineur dans les pathologies humaines de cette région ; ces résultats sont corroborés par l’absence, durant ces dernières décennies, de cas documentés d’infection aigüe due à SFSV en Europe occidentale. Nous avons donc pu concentrer notre travail sur le deuxième groupe de phlébovirus d’intérêt chez l’homme, le groupe des Sandfly fever Naples virus, qui inclut notamment TOSV. Nous avons démontré l’existence d’un lien épidémiologique entre les infections à Leishmania infantum et celles à TOSV, certainement dû au fait que ces pathogènes sont transmis par un vecteur commun (Phlebotomus perniciosus). Les analyses statistiques ont montré que les personnes exposées aux infections à TOSV ont plus de chance d’être aussi infectées par les parasites leishmanies (et vice versa). En admettant que ce lien épidémiologique entre leishmanioses et infections à TOSV est représenté par l’exposition à la piqûre d’un vecteur commun, cette étude confirme l’implication de Phlebotomus perniciosus en tant que vecteur principal de TOSV dans le sud de la France. Cette étude suggère également que certaines données épidémiologiques disponibles pour la leishmaniose pourraient être utilisées pour décrypter l’épidémiologie des infections à TOSV.La deuxième partie de cette thèse est consacrée à la détection, l’isolement et la caractérisation de virus, déjà connus ou inconnus, dans les populations de phlébotomes en France et en Afrique du Nord. Pour atteindre cet objectif, nous avons dû développer une plateforme d’analyse à au débit, adaptée pour la découverte de virus dans les phlébotomes, qui permette de traiter un grand nombre d’échantillons à faible coût. Cette plateforme a récemment été complétée par un outil de Next Generation Sequencing, afin de réaliser la caractérisation génétique complète des virus isolés et découverts. Au total, 12 576 phlébotomes ont été capturés au cours de 12 campagnes de capture menées en France, en Tunisie et en Algérie. Au sein d’une même zone géographique, la découverte de plusieurs nouveaux phlébovirus, ainsi que leur taux d’infection observé dans les populations de phlébotomes, ont démontré que la diversité de phlebovirus est bien plus importante qu’attendue.Dans la troisième partie de cette thèse, une étude de séroprévalence a été menée sur des sérums humains en utilisant des tests comparatifs de neutralisation de virus. Cette étude nous a permis d’exclure le virus Punique, récemment découvert, de la liste des principales menaces en santé publique au nord de la Tunisie, et de confirmer que TOSV est le principal phlebovirus pathogène ayant un impact en santé publique dans cette région du pays. Cette méthode de neutralisation est capable d’identifier précisément, parmi des virus génétiquement proches, le virus contre lequel les anticorps présents dans le sang ont été produits, ce qui permet de déterminer la capacité de chacun de ces virus à jouer un rôle en santé publique. / Sandflies are vectors of various arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae, and of parasites in the genus Leishmania. Human diseases caused by infection with sandfly-borne phleboviruses are known for a long time, but they remain neglected due to the lack of epidemiological knowledge and of diagnostic tools.The first part consisted of seroprevalence studies in human sera to address the public health impact in south-eastern France of two recognized sandfly-borne phleboviruses, namely Toscana virus (TOSV) and Sandfly fever Sicilian virus (SFSV). Concerning the latter, specific IgG were detected in less than 1% of tested sera, suggesting that SFSV play a minor role in human disease in the region; this finding was corroborated with the lack of documented case of acute infection due to SFSV in Western Europe during the last decade. This pleaded for focusing on the other group of sandfly fever viruses known for their human interest, namely the group of Sandfly fever Naples virus that includes TOSV. We demonstrated an existing epidemiological relationship between Leishmania infantum and TOSV infections, presumably through the transmission by the common arthropod vector (Phlebotomus perniciosus). Statistical analysis showed that persons exposed to TOSV infection are at greater risk of being infected with Leishmania parasite (and vice versa). Assuming that epidemiological link between leishmaniasis and TOSV infection may be represented by the exposure to the bite of a common vector, this study confirms the involvement of Phlebotomus perniciosus as the major vector of TOSV in the South of France. This study also suggests that some of the epidemiological data available on Leishmaniasis may be used to decipher the epidemiology of TOSV infections..The second part of this thesis was dedicated to detection, isolation and characterization of existing and/or new phleboviruses in sandfly populations in France and in North Africa. To achieve this aim, we had to set up a high-throughput cost-effective platform amenable to virus discovery in sandflies; this sandfly-processing platform has been recently docked to a Next Generation Sequencing platform for full genetic characterization of newly isolated and discovered viruses. A total of 12,576 sandflies were trapped during 12 campaigns conducted in France, Tunisia and Algeria. The discovery of several new phleboviruses and their observed frequency in sandfly populations has clearly demonstrated that within a given geographic area, virus diversity is much higher than previously believed.In the third part of this thesis, a seroprevalence study based on comparative virus neutralization tests was performed on human sera and allowed to exclude the newly described Punique virus from the list of major public health threats in northern Tunisia, and to confirm that TOSV is the dominant phleboviral pathogen with an impact on public health in this part of the country. This neutralization method is suitable to identify precisely the virus against which antibodues were elicited, allowing to discriminate among closely related phleboviruses, and to determine their propensity to play a role in public health.

Parmi les phlébovirus (famille des Bunyaviridae, genre Phlebovirus), ceux qui sont transmis par les phlébotomes de l'Ancien Monde sont largement distribués dans le bassin méditerranéen. Les infections humaines causées certains de ces phlébovirus sont connues depuis longtemps, mais elles restent tout de même négligées en médecine en raison de l'absence de données épidémiologiques solides (problème des réactions croisées) et d'outils de diagnostic rapides et fiables.La première partie de cette thèse a été consacrée à l'optimisation d'un test de neutralisation du virus pour étudier la séroprévalence de 5 virus, et leur capacité respective à infecter les humains et les animaux.La deuxième partie visait à mesurer la séroprévalence de phlébovirus appartenant aux 3 complexes antigéniques transmis par les phlébotomes dans le bassin méditerranéen (Sandfly fever Naples, Sandfly fever Sicilian et Salehabad). Ces études ont été menées sur des sérums de chiens et de chats en Tunisie, Portugal, Grèce/Chypre.La troisième partie a montré la capacité de virus récemment découverts dans le serocomplexe Salehabad (Adana et Medjerda valley virus) à infecter l'homme et les animaux traduisant un potentiel pathogène à explorer par des études spécifiques.La dernière partie a démontré la présence du virus Toscana en Kabylie (Algérie du Nord), et l'exposition extrêmement élevée des populations humaines vivant dans la région, avec des prévalence 10 fois plus élevées que dans les régions les plus à risque du sud-est de la France. / Sandfly-borne phleboviruses, transmitted by phlebotomine sandflies and belonging to the genus Phlebovirus within the Bunyaviridae family are widely distributed in Mediterranean basin. Human diseases caused by infection with phleboviruses are known for a long time, but they are still neglected due to the lack of epidemiological knowledge and of diagnostic tools.The first part of this thesis was dedicated to optimize a comparative virus neutralisation test to study the seroprevalence of selected phleboviruses and to assess the capacity of each virus to infect humans and animals. The second part aimed to estimate the epidemiology of phlebovirus serocomplexes (Naples, Sicilian and Salehabad) in Mediterranean basin. In order to update the presence of these viruses and their capacity to infect animals, several serologic studies were carried out on animal blood samples in Tunisia, Portugal, Greece and Cyprus. The results demonstrated that the phleboviruses belonging to 3 distinct groups are widely circulating and capable to infect non human vertebrate at different rates in studied countries.The third part showed the capacity of newly discovered viruses (Adana and Medjerda valley viruses) belonging to Salehabad serocomplex to infect human and animal at low and high rates, respectively. These findings suggest the medical and veterinary importance of these viruses. The last part of this thesis, confirm the circulation of Toscana virus by seroprevelance study which was carried out in local population in north Algeria where Toscana virus was isolated recently. The high rate of circulate suggests that Toscana virus is heavily affecting sandfly-exposed people in Algeria.

The Adames strain of Punta Toro virus (PTV-A) causes acute hepatic disease in hamsters and mice similar to that seen in natural Rift Valley fever virus (RVFV) infection, while the Balliet strain (PTV-B) is apathogenic. The ability of PTV-A to suppress the interferon (IFN) response has been demonstrated in hamsters and is thought to be a contributing factor to PTV-A's pathogenicity in hamsters. PTV-B is not assumed to exhibit this IFN-antagonistic activity, as it stimulates production of significantly higher IFN-β levels. To elucidate the role of IFN in resistance of mice to PTV-B infection, we utilized mice deficient in a critical IFN signaling protein, STAT-1. We found that these mice were drastically more susceptible to PTV-B, which caused 100% lethality compared to 0% in their wild-type counterparts. STAT-1 deficient mice were also more susceptible to PTV-A, as these mice succumbed to infection significantly earlier than wild-type mice (p=0.0058). We sought to determine whether PTV-A's IFN-antagonistic mechanism is functional in mice by examining expression of IFN-β in primary macrophages infected with either strain. We found that IFN-β protein concentration is higher in samples taken from PTV-B-infected cells. We employed quantitative PCR arrays specific to IFN signaling and response pathways to evaluate changes in gene expression throughout the course of infection with either virus strain. We found several genes with differentially regulated expression between PTV-A- and PTV-B-infected macrophages, including Ifnβ1 and multiple Ifnα subtypes. Also, several genes coding for inflammatory and chemotactic molecules, Cxcl11, Cxcl10, Cxcl9, Vcam1, and Il6, demonstrated increased expression in PTV-B samples compared to PTV-A. Of particular interest, Isg20, a 3'-5' exonuclease with specificity for single-stranded RNA, was stimulated ~2-fold higher by PTV-B, and Iigp1, from the family of GTPases associated with host defense against intracellular pathogens, was stimulated ~2.7-fold higher by PTV-B. The individual functions of each of these genes in mouse resistance to PTV-B could be a focus of future studies to better understand essential host defense mechanisms to phleboviral infection.

Le virus Toscana : épidémiologie en région PACA-Corse et interaction avec la réponse immunitaire innée Le Toscana Virus (TOSV) est un arbovirus (arthropod-borne virus) qui appartient au genre Phlebovirus (famille des Bunyaviridae). Il est transmis par la piqûre d'un insecte hématophage, le phlébotome (Phlebotomus perniciosus et P. perfiliewi), piquant notamment l'homme. Le vecteur P. perniciosus est présent en France en forte densité sur le littoral méditerranéen. TOSV est endémique en France (isolé à partir de phlébotomes ou de sujets contaminés) et dans de nombreux pays frontaliers. TOSV est associé à des infections humaines généralement bénignes spontanément résolutives, la plupart des infections étant asymptomatiques ou pauci-symptomatiques. Toutefois, dans certains cas, le virus est à l'origine d'inflammations graves du système nerveux central (méningites ou méningo-encéphalites), nécessitant une hospitalisation. TOSV est ainsi l'un des agents viraux les plus fréquemment associés aux infections neuroméningées chez l'homme d'avril à octobre, lorsque les conditions météorologiques sont les plus favorables à l'activité du vecteur. Ce travail aborde deux aspects de l'infection à TOSV : un aspect épidémiologique et un aspect fondamental sur la relation entre l'immunité innée cellulaire et TOSV. L'étude épidémiologique est la première réalisée dans les régions PACA-Corse chez les donneurs de sang. Celle-ci a mis en évidence une prévalence IgM/IgG de 14% (allant de 7,8 % dans la Hautes Alpes à 18,8% dans les Alpes Maritimes). La prévalence des IgM est de 3,3% pour l'ensemble des départements, avec la valeur la plus élevée pour la Corse (8,7%). / Toscana Virus (TOSV) is an arbovirus (arthropod-borne virus) which belongs to the genus Phlebovirus (family Bunyaviridae). It is transmitted to humans by the bite of a blood sucking sandfly (Phlebotomus perniciosus and P. perfiliewi). The vector P. perniciosus is present in high densities in France on the Mediterranean coast. TOSV is endemic in France (isolated from sandflies or contaminated patients) and in neighbouring countries. When weather conditions are most favorable to the vector activity (April-October), TOSV causes asymptomatic or mild (flu-like symptoms) infections in humans. However, in some cases, the virus can be can be responsible for a severe inflammation of the central nervous system (CNS) resulting in meningitis or meningoencephalitis, requiring hospitalization. This work focused on two aspects of TOSV infection: the epidemiology of the virus in the south area of France and the relationship between innate immunity and TOSV. The epidemiological study was the first conducted in the PACA-Corse area among blood donors. Our results showed a mean seroprevalence of 14% IgM / IgG ranging from 7.8% in the Hautes Alpes to 18.8% in the Alpes Maritimes. The mean IgM seroprevalence was 3.3%, with the highest value for Corsica (8.7%). The results demonstrated that a large population of blood donors was once infected with TOSV, suggesting a potential transfusion risk. The second study focused on a more fundamental aspect of TOSV infection: the interaction between the virus and the interferon-β response (IFN-β). We demonstrated that TOSV replication is sensitive to IFN-β treatment but the virus is able to inhibit the induction of IFN-β.

Les phlébovirus présentent sont présents dans toutes les régions du globe. Certains phlébovirus transmis par phlébotomes provoquent une maladie fébrile et des infections du système nerveux central. Depuis, de plus en plus de données montrent que la péninsule des Balkans joue un rôle majeur dans l'émergence de maladies à transmission vectorielle. Au début de ce travail, on comptait un nombre très limité de phlébovirus identifiés et isolés dans cette région. Une étude intégrée et transdisciplinaire en vue d'un inventaire des virus circulant dans pays des Balkans. (i) Un total de 3,850 phlébotomes sont été recueillis dans sept pays des Balkans en 2014 et 2015. Ils ont été testés pour la présence d'ARN viral et inoculé sur des cellules VERO afin d'isoler le virus détecté; (ii) des études de séroprévalence utilisant des tests de neutralisation ont été effectuées sur des échantillons de bovins et de moutons pour évaluer à deux agents pathogènes humains : le virus Toscana (TOSV) et le virus Sandfly fever Sicilian virus (SFSV). Nos résultats se composent de (i) la découverte et le séquençage de 3 nouveaux phlébovirus appartenant à 2 espèces différentes, (ii) la première identification du genotype B de TOSV en Croatie, (iii) la preuve de la co-circulation de deux genotypes (B et C) de TOSV, (iv) des taux d'anticorps neutralisants qui sont beaucoup plus élevés chez les bovins et les moutons pour le SFSV que pour TOSV. En conclusion, les résultats obtenus au cours de ce travail démontrent qu’es les Balkans représentent une zone de très importante activité pour les phlebovirus et donc mérite une surveillance particulière à cause du risque d’émergence et de dissémination. / Phleboviruses have a worldwide distribution. In the areas where sand flies are present, some of the sandfly-borne phleboviruses cause febrile illness and central nervous system infections. Sandfly fever was first reported in the Balkan Peninsula at the end of the 19th century. Since there is accumulating data showing that the Balkan peninsula plays a major role in the emergence of vector-borne diseases. At the outset of this work, a very limited number of phleboviruses had been identified and isolated in this region. To fill this gap, an integrated and transdisciplinary study was designed aiming at an inventory of viruses circulating in Balkans and associated seroprevalence studies using domestic animals: (i) a total of 3,850 sandflies were collected in seven Balkan countries (Albania, Bosnia-Herzegovina, Croatia, Kosovo, Montenegro, Republic of Macedonia and Serbia) in 2014 and 2015. They were tested for the presence of viral RNA and inoculated on VERO cell for virus isolation; (ii) seroprevalence studies using neutralisation tests were performed on cattle and sheep samples to assess the level of exposure to two human pathogens, Toscana virus (TOSV) and Sandfly fever Sicilian virus (SFSV). Our results consist of (i) the discovery and sequencing of 3 novel phleboviruses belonging to 2 different species, (ii) the identification for the first time of TOSV lineage B in Croatia, (iii) evidence of co-circulation of two lineages (Lineage B and C) of TOSV, (iv) rates of neutralising antibodies that are much higher in cattle and sheep for SFSV than for TOSV. Together the findings obtained during this work demonstrate that the Balkan area is a hot spot for phleboviruses.

Emerging arthropod-borne viruses, such as alphaviruses and bunyaviruses, represent a serious threat to human and animal health worldwide, and for most of them, vaccines and specific treatments are unavailable. Viral host cell entry can be divided into several entry checkpoints, and the most important checkpoints for low pH-dependent enveloped viruses, such as bunyaviruses and alphaviruses, include receptor binding at the cell surface and, followed by endocytosis, low pH dependent membrane fusion from within intracellular compartments. A more thorough understanding of the detailed mechanisms allowing the viruses to pass these checkpoints is a pre-requisite for the design of viral entry inhibitors. This thesis reports the in vitro analysis of native alphavirus-receptor interactions, with the help of electron cryo-microscopy and icosahedral reconstruction of virus-recaptor complexes, using the prototypic alphavirus Semliki Forest virus (SFV) and the C-type lectin DC-SIGN. Together with results from collaborative work on SFV glycosylation, this study provides progress in defining the binding sites of DC-SIGN at the surface of SFV. Second, an in vitro system for phlebovirus fusion was developed using standard fluorometry, and has been characterized with the help of electron cryo-microscopy. It was discovered that negatively charged phospholipids with a conical shape, including the late endosomal phospholipid BMP, allow efficient phlebovirus fusion in vitro, thereby providing a possible rationale for phlebovirus fusion in late endosomes. Furthermore, electron cryo-microscopy of phlebovirus-liposome complexes allowed the capture of early stage fusion intermediates and laid the basis for possible future higher resolution studies of these fusion intermediates.

Circulation des Phlébovirus en Turquie, l'Iran et l'Algérie a été étudiée. L'isolement, la caractérisation génomique, les relations phylogénétiques de six virus ont été présentées: le virus Adana (ADAV), deux souches de virus Toros (TORV), le virus Zerdali (ZERV) de la Turquie; le virus Dashli (DASHV) de l'Iran; le virus Toscana (TOSV) d'Algérie. Cette étude a commencé avec la collection de 38,131 phlébotomes de la nature. La méthode de séquençage de nouvelle génération (NGS) à haut débit nous à été utilisée pour l’analyse des génomes complet des virus isoles. En conclusion, cette étude a d'importantes contributions sur phlébovirus négligées. Voici quelques-unes des contributions significatives; (i) ZERV et TORV qui sont étroitement apparentés au virus Tehran (THEV) et le virus Corfou (CFUV), respectivement, ont été isolés depuis 56 et 30 ans des premiers isolements de THEV et CFUV, respectivement, (ii) Détection du virus ADAV un animal domestique et sur quelques sérums humain par test de neutralisation. Ce virus ADAV constitue avec le virus le virus (SALV), le virus Arbia (ARBV), et le virus (ADRV) le groupe Salehabad. Seul le virus ADRV a été détectée dans le liquide cérébro-spinal auparavant landais que avec les autres, aucune preuve pathogène n’a été détectée, (iii) Nous avons découvert la plus récente circulation phlébovirus en Iran après 56 années, (iv) TOSV a été isolé en Algérie pour la première fois et la circulation a été confirmée par séropositivités dans le sérum humain. / Sandfly-borne phlebovirus circulation in Turkey, Iran, and Algeria was investigated. The isolation, genomic characterization, phylogenetic relationships of 6 viruses was presented: Adana virus (ADAV), two strains of Toros virus (TORV), Zerdali virus (ZERV) from Turkey; Dashli virus (DASHV) from Iran; Toscana virus (TOSV) from Algeria. This study has begun with the collection of 38,131 sandflies from nature. The well established, high-throughput methodology was applied for the discovery of viruses including PCR tools and cell culture methods. Next generation sequencing (NGS) technology facilitated to perform complete genome analysis of the isolated viruses. In conclusion, this study has contributions to the neglected sandfly-borne phlebovirus group and filled some gaps about the circulation of these agents in Turkey, Iran, and Algeria. Following are some significant contributions; (i) ZERV and TORV which are closely related to Tehran virus (THEV) and Corfou virus (CFUV), respectively were isolated after 56 and 30 years of the first isolations of THEV and CFUV, respectively, (ii) There was no evidence of the pathogenicity of Salehabad virus (SALV) and Arbia virus (ARBV) except the detection of Adria virus (ADRV) in CSF until ADAV which belongs to the Salehabad serocomplex was detected in domestic animal and very few human sera by neutralization assay, (iii) We have discovered the most recent sandfly-borne phlebovirus circulation in Iran after 56 years, (iv) TOSV was isolated in Algeria for the first time and circulation was confirmed by seropositivities in human sera.

Submitted by PPG Zoologia (zoologia-pg@pucrs.br) on 2018-08-01T18:22:00Z No. of bitstreams: 1 Almeida MAB___TESE___VERS?O FINAL.pdf: 2953118 bytes, checksum: 99cbefa9c38c7969abce4bafc4b20d54 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-08-02T17:55:11Z (GMT) No. of bitstreams: 1 Almeida MAB___TESE___VERS?O FINAL.pdf: 2953118 bytes, checksum: 99cbefa9c38c7969abce4bafc4b20d54 (MD5) / Made available in DSpace on 2018-08-02T18:39:41Z (GMT). No. of bitstreams: 1 Almeida MAB___TESE___VERS?O FINAL.pdf: 2953118 bytes, checksum: 99cbefa9c38c7969abce4bafc4b20d54 (MD5) Previous issue date: 2018-03-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Nonhuman primates (NHP) are susceptible to many arboviruses, including the yellow fever (YF) virus. Although native to Africa, this virus found susceptible NHP and competent mosquito vectors for maintaining its transmission in American forests. A high sensitivity of NHP to YF led health agencies to monitor these animals as a way of monitoring the disease in Brazil. The State of Rio Grande do Sul (RS) began this surveillance in 2002, which has detected the arboviruses Oropouche and Saint Louis (SLEV) and a YF epizootic that killed more than 2,000 NHP (Alouatta caraya and A. guariba clamitans) between 2008 and 2009. The objectives of this PhD thesis research were to generate models of niche suitability for YF based on that epizootic and prospect arboviruses in NHP in northwestern RS. The maximum entropy algorithm - Maxent was used to generate distribution models of Alouatta spp. and the mosquito vector Haemagogus leucocelaenus. Together with climatic, topographic and vegetative variables, these models served as predictor layers to model the occurrence of the disease based on the points of death of NHP of YF. The most influential variables in the YF models were the variation in air humidity, distribution of Alouatta spp. and maximum wind speed followed by mean annual rainfall and maximum temperature. Therefore, support for the influence of the rainfall regime and the ambient temperature on the cycle of jungle YF was found. Wind speed and direction can play an important role in the dispersal of infected mosquitoes and, consequently, the virus. The models based on the occurrence of dead NHP in the first months of the epizootic identified suitable areas to where the disease spread a few months later. In addition, 19 arboviruses were prospected in 40 blood (viral isolation and PCR) and serum (hemagglutination inhibition and neutralization tests [NT]) samples collected from 26 black howler monkeys (A. caraya) belonging to three populations in four field campaigns in the municipality of Santo Ant?nio das Miss?es, RS, between 2014 and 2016. There was no detection of circulating virus, but antibodies to Flavivirus SLEV and Ilh?us and Phlebovirus Icoaraci was found by NT. Evidence of the contact with Ilh?us and Icoaraci are the southernmost records in Brazilian NHP. An increase in antibodies to SLEV detected between two consecutive captures of the same individual is compatible with a recent contact with the virus. An adult male captured in one of the areas presented concomitant infection by the Oropouche, SLEV and YF viruses by NT. Further studies are necessary to understand the role played by NHP and other vertebrates in the circulation of arboviruses in the region, to assess potential risks to NHP and public health, and to identify the driving forces responsible for the dispersal of the YF virus during epizootics in wildlife populations. / Os primatas n?o-humanos (PNH) s?o suscet?veis a diversos arbov?rus, incluindo o v?rus da febre amarela (FA). Embora origin?rio da ?frica, esse v?rus encontrou PNH suscet?veis e mosquitos vetores competentes para sua transmiss?o em matas nas Am?ricas. Uma alta sensibilidade dos PNH ? FA levou ?rg?os de sa?de a monitorar esses animais como forma de vigiar a doen?a no Brasil. O Estado do Rio Grande do Sul (RS) iniciou essa vigil?ncia em 2002, a qual detectou os arbov?rus Oropouche e Saint Louis (SLEV) e uma epizootia de FA que matou mais de 2000 PNH (Alouatta caraya e A. guariba clamitans) entre 2008 e 2009. A presente tese de doutorado teve como objetivos gerar modelos de adequabilidade ambiental para FA com base nessa epizootia e prospectar arbov?rus em PNH no noroeste do RS. Foi utilizado o algoritmo de m?xima entropia ? Maxent para gerar modelos de distribui??o de Alouatta spp. e do mosquito vetor Haemagogus leucocelaenus. Esses modelos serviram como camadas preditoras para, junto a vari?veis clim?ticas, topogr?ficas e vegetacionais, modelar a ocorr?ncia da doen?a baseada nos pontos de morte de PNH por FA. As vari?veis mais influentes nos modelos da FA foram a varia??o na umidade do ar, a distribui??o de Alouatta spp. e a velocidade m?xima dos ventos, seguidas pela precipita??o m?dia anual e a temperatura m?xima. Portanto, foi confirmado suporte para a influ?ncia do regime de chuvas e da temperatura ambiente no ciclo da FA silvestre. A velocidade e a dire??o do vento devem desempenhar um importante papel na dispers?o de mosquitos infectados e, consequentemente, do v?rus. Os modelos baseados na distribui??o espacial de PNH mortos nos primeiros meses da epizootia identificaram ?reas adequadas para onde a doen?a avan?ou poucos meses mais tarde. Tamb?m foram prospectados 19 arbov?rus em 40 amostras de sangue (isolamento viral e PCR) e soro (inibi??o da hemaglutina??o e testes de neutraliza??o [NT]) coletadas em quatro campanhas de campo entre 2014 e 2016 de 26 bugios-pretos (A. caraya) de tr?s popula??es no munic?pio de Santo Ant?nio das Miss?es, RS. N?o houve detec??o de v?rus circulante, mas sim de anticorpos para os Flavivirus SLEV e Ilh?us e o Phlebovirus Icoaraci por NT. As evid?ncias de contato com Ilh?us e Icoaraci s?o as primeiras em PNH no extremo sul do Brasil. Um aumento de anticorpos para SLEV detectado entre duas capturas consecutivas do mesmo indiv?duo ? compat?vel com um contato recente com o v?rus. Um macho adulto capturado em uma das ?reas apresentou infec??o concomitante pelos v?rus Oropouche, SLEV e FA por NT. Mais estudos s?o necess?rios para compreender o papel de PNH e outros vertebrados na circula??o de arbov?rus na regi?o, avaliar poss?veis riscos para PNH e a sa?de humana e identificar as for?as motrizes respons?veis pela dispers?o do v?rus da FA durante epizootias em popula??es selvagens.