Les huiles essentielles de citronnelle (Cymbopogon citratus), de géranium (Pelargonium graveolens) et de vétiver (Vetiveria zizanioides) sont utilisées partout dans le monde pour leur activité répulsive contre les principaux vecteurs (moustiques, tiques) de maladies infectieuses chez l'Homme (paludisme, chikungunya, …). L'application cutanée de ces produits naturels pour éviter le contact avec un vecteur n'avait pas été encore envisagée comme moyen de limiter les premiers stades de l’infection par l'agent pathogène transmis par le vecteur. Pour vérifier cette hypothèse, les travaux ont été consacrés à la mise en place d'un cadre structuré pour la réévaluation chimique et biologique des trois huiles essentielles sur le modèle du virus du Ross River (alphavirus) de la même famille que le virus du Chikungunya. La caractérisation chimique des huiles essentielles avec une technique de haute résolution (GC×GC/TOF-MS) a permis d'établir leur profil chémotypique précis. L'utilisation de marqueurs spécifiques (clones moléculaires du virus) a permis d'établir l'inhibition de la réplication virale en fonction des conditions d'application des huiles essentielles de géranium et citronnelle. Ces résultats suggèrent l'intérêt d’une huile essentielle répulsive dans les premiers stades d'une infection par un vecteur. À ce titre, l'étude comparative établit la haute valeur ajoutée de l'huile essentielle de géranium et oriente la recherche de nouveaux anti-infectieux naturels vers des complexes riches en monoterpènes. / Essential oils of citronella (Cymbopogon citratus), geranium (Pelargonium graveolens) and vetiver (Vetiveria zizanioides) are used worldwide as topical repellent against the main vectors (mosquitoes, ticks) of human infectious diseases (Malaria, chikungunya, …). Skin treatment with these natural products, initially to avoid contact with the vector had not yet been considered as a way to disrupt the early stages of infection when the repelling action fails. To check this hypothesis, a structured framework has been performed for the chemical and biological re-evaluation of the three essential oils. The latter was tested against Ross River virus (alphavirus) that belongs to the same family of Chikungunya virus. Analysis of essential oils using a high-resolution technique (GC × GC / TOF-MS) resulted in a more accurate chemotypical profile of the local production. The use of specific markers (molecular clones of the virus, Saclay CEA) allowed to establish the inhibition of viral replication depending of the conditions of geranium and citronella essential oils application. These results suggest the great interest of an essential oil topical repellent in the early stages of a vector infection. The comparative study established the high value of geranium essential oil and gave future direction to the discovery of new anti-infectious solutions from monoterpenes-rich natural complexes.

Chikungunya est un alphavirus transmis par les moustiques (Aedes) et qui provoque de la fièvre, des éruptions cutanées, des myalgies et des arthralgies. La maladie (CHIKVD) est transitoire, mais des formes sévères menant à des arthrites chroniques incapacitantes ont été signalées. Nous avons dans un premier temps étudié prospectivement les paramètres cliniques et immunologiques associés à la maladie chez des patients hospitalisés et identifiés comme étant 'guéris' ou 'chronique' à M12 après l'infection. Dans la deuxième partie, nous avons observé in vitro les mécanismes et le rôle de l'apoptose dans le processus infectieux permettant au virus de persister dans les sanctuaires tissulaires. En phase aiguë, une forte réponse immune dominée par une activation des cellules NK/dendritique/cellules T, la production d'anticorps spécifiques et une faible production de cytokines Th1 > Th2 a été observée mais sans aucune différence significative entre les deux groupes. Cependant, la virémie initiale s'est révélée beaucoup plus élevée dans le groupe chronique est nous avons pu identifier du matériel viral dans les macrophages du tissu synovial d'un patient chronique post-CHIKVD (M18). Dans la deuxième partie de l'étude, nous avons constaté que CHIKV est capable d'induire l'apoptose par la voie intrinsèque et extrinsèque et également par un mécanisme 'bystander'. De plus, nous avons observé que le CHIKV présent dans des corps (blebs) apoptotiques était capable d'infecter les cellules voisines (Hela et macrophage MM6). Notre étude a permis de mettre en évidence pour la première fois que CHIKV contrôle et détourne à son profit les mécanismes de défense anti-infectieux.

Single-stranded positive-sense RNA viruses commonly rearrange host cell organelle membranes into neo-organelles which are involved in virus replication and assembly. These organelles serve to concentrate viral and host factors as well as to conceal viral RNA replication activities from host cell surveillance. To date, many virus-induced membrane rearrangements have been studied by targeted electron tomographic (ET) imaging of specific viral structures at timepoints of known interest. However, the broad cellular context within which these membrane modifications occur and how they change over time are not well understood. A question spanning many virus families is the morphological mechanism of formation of membrane rearrangements. Additionally, it is largely unknown how the membrane modifications affect the morphology of the organelle of origin. In this study, we address specific questions about virus-derived organelles induced by two positive-sense RNA viruses: the coronavirus mouse hepatitis virus (MHV) and the alphavirus Venezuelan equine encephalitis virus (VEEV). Utilizing serial sectioning and montage imaging for ET, volumes representing approximately 10% of virus-infected cells were imaged and detailed organelle analysis was performed. Using MHV-infected cells, we demonstrate that coronavirus-induced double-membrane vesicles (DMVs) are formed by budding from the endoplasmic reticulum (ER) and are trafficked to lysosomes for degradation. The ER remains largely morphologically normal early in infection despite the presence of hundreds of DMVs; however, late in infection, virus envelopment in the ER lumen leads to loss of cisternal morphology. For the alphavirus VEEV, we analyze the structure and origin of virus-derived cytopathic vacuoles II (CPVII). We identify four distinct morphological forms of CPVII and provide evidence that all four forms are derived from the Golgi apparatus. Additionally, a protocol is outlined for a newly-developed method for improved cell ultrastructure during genetically-encoded peroxidase tagging of membrane-proteins. This method is also amenable to ET. Overall, this work provides morphological cellular context for virus-induced membrane rearrangements from two families of positive-sense RNA viruses. Analysis of virus-host cell interactions from this large-scale ultrastructural perspective has the potential to lead to new approaches and strategies to combat current and future viral diseases.<br>

No description available.

Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy. / Doctor of Philosophy / Alphaviruses are arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that infect millions of people annually via the bite of female mosquitoes. These viruses are major public health threats due to their ability to infect humans and animals and infections resulting in a range of debilitating diseases. Viruses within this genus are clustered into two groups: Old World and New World, based on geographical origin and distribution. While New World alphaviruses are known for inducing severe encephalitis (i.e., swelling in the brain), a hallmark symptom of the Old World alphaviruses is the development of incapacitating polyarthralgia (i.e., widespread joint pain) that can persist for years following initial infection. To date, the most effective means of combatting these viruses is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens. Given the close genetic relationship between chikungunya virus (CHIKV) and Mayaro virus (MAYV), our research has focused on harnessing cross-reactive immunity between these emerging alphaviruses. We discovered this cross-reactivity provides protective immunity to both viruses (i.e., CHIKV and MAYV) after exposure to only one (i.e., CHIKV) of the viruses. Next, we characterized the potency of a small, single-domain antibody (sdAb) to neutralize a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express this sdAb to target both CHIKV and MAYV within this deadly mosquito vector. These findings are particularly significant because they provide the foundation for a novel approach to controlling and preventing outbreaks of these emerging alphavirus pathogens that obliterate quality of life in public health settings around the globe.

The world is grappling with an escalating risk of viral outbreaks of pandemic proportion, with zoonotic RNA viruses such as chikungunya virus (CHIKV) and SARS-CoV-2 posing significant threats to global health. Several environmental and evolutionary factors have fueled the emergence and spread of infection, creating a constant arms race against emerging pathogens. Current prevention and mitigation strategies are inadequate, necessitating tools to prevent and control viral infections; innovative strategies are needed in the pipeline to address significant challenges. CHIKV is a mosquito-borne virus that has caused millions of disease cases worldwide and is a reemerging threat with increasing potential to become endemic in the US. Currently, there are no licensed treatments available to protect against CHIK disease, making the development of a vaccine crucial. Live-attenuated vaccines (LAVs) have traditionally been a promising strategy due to their high immunogenicity and cost-effectiveness. However, concerns regarding adverse side effects and the potential for viral replication leading to pathogenic reversions or transmission into mosquitoes have limited their use. To that end, we have developed a new generation of safer vaccines by modifying the standard LAV platform through innovative attenuating strategies. Our dual-attenuated platform utilizes a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) as a vaccine backbone which expresses antiviral mouse cytokines IFN-γ or IL-21, as an additional mechanism to control infection. In several mouse models, both cytokine-expressing candidates showed reduced footpad swelling and minimal to no systemic replication or dissemination capacity compared to the parental vaccine post-vaccination. Importantly, these candidates conferred full protection from wildtype CHIK disease. Our IFNγ-expressing vaccine showed the most significant attenuation of viral replication. To understand the underlying mechanism, we identified three IFNγ-regulated antiviral genes (Gbp1/2 and Ido1) that were highly upregulated in 3T3 mouse fibroblasts post-infection with the IFN-γ-expressing candidate but not the parental backbone. To further investigate the role of these genes in restricting viral replication and enhance the clinical relevance of our vaccine platform, we redesigned our vaccine to express human IFNγ (hIFNγ) and performed viral growth kinetics in MRC5 human lung fibroblasts. Our vaccine showed reduced viral replication compared to controls and high expression of human GBP1/2/3 was observed post-infection. Overexpression of these genes demonstrated a direct impact on viral replication against wildtype CHIKV. These findings shed light on the mechanism of action of our vaccine and highlight the potential of targeting IFNγ-regulated antiviral genes for developing effective vaccines against CHIKV. Our results provided a foundation for investigating the broad-use application of IFN-γ against other alphaviruses for vaccine or therapeutic design. We evaluated the effects of increasing levels of exogenous hIFNγ on Mayaro virus (MAYV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV). We observed a positive dose-dependent relationship between hIFNγ and decreasing viral titers for all three viruses. Interestingly, we also observed similar patterns of GBP upregulation with MAYV and RRV, both Old World alphaviruses, but not with VEEV, a New World alphavirus. This finding may indicate an alternative IFNγ-stimulated pathway responsible for controlling different alphaviruses. Overall, these studies establish a fundamental role of IFNγ in controlling viral infection and highlight its potential use in both vaccine and therapeutic intervention. While LAVs are a gold standard for developing immunity against a virus, the urgency of responding to an active and deadly pandemic has promoted the use of faster strategies such as mRNA vaccines. Once the viral sequence was known, these vaccines were comparatively quick to produce for SARS-CoV-2 and prevented millions of disease cases at the height of their introduction. However, the emergence of variants of concerns bypassing previous immunization efforts has demonstrated the need for complementary treatments such as antivirals to control disease. To that end, we evaluated several rhodium organometallic complexes as potential antivirals against SARS-CoV-2. We show that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(ICy)Cl2 and Cp*Rh(dpvm)Cl are non-toxic in Vero E6 and Calu3 cells and reduce SARS-CoV-2 plaque formation up to 99%. These complexes have previously demonstrated high antimicrobial activity against multiple antibiotic-resistance bacteria and with our results, support their potential application as pharmaceuticals, warranting further investigation into their activity. / Doctor of Philosophy / The global response to the COVID-19 pandemic, and its far-reaching impact, revealed significant shortcomings in public health preparedness for emerging viruses. Despite efforts to develop vaccines and antivirals to prevent and treat disease, current mitigation strategies have proven insufficient to eradicate the pathogen. The emergence of viral outbreaks caused by viruses such as chikungunya (CHIKV) and SARS-CoV-2 underscores the ongoing threat posed by emerging infectious diseases. Improved countermeasures are urgently needed to address gaps in vaccine and antiviral development. CHIKV is a mosquito-borne virus that has caused millions of infections across hundreds of countries with the emergent potential to become endemic in the US. Currently, there are no vaccines available to the public; therefore, it is important to generate and administer an effective vaccine before further spread of the virus. To this end, we developed innovative live-attenuated vaccines (LAVs) against CHIKV using a weakened chimeric backbone of CHIKV and its close relative, Semliki Forest virus (SFV), along with vaccine-driven expression of antiviral cytokines to control viral replication. Vaccination of highly susceptible mice with these cytokine-expressing vaccines produced significantly decreased side-effects compared to the parental virus not expressing the cytokines. Additionally, these viruses had significantly restricted viral replication capabilities while robustly protecting mice from a semi-lethal CHIKV infection. Our interferon-gamma (IFNγ) expressing vaccine had the greatest impact on viral replication, and we investigated the mechanism leading to this attenuation. To assess the clinical relevance of our vaccine platform, we redesigned the virus to express human IFNγ and identified a specific pattern of IFNγ-stimulated genes that are potentially responsible for limiting CHIKV replication. Furthermore, we demonstrated the broad therapeutic use of IFNγ against other medically relevant alphaviruses. Overall, these studies establish an improved mechanism to create safer vaccines without compromising efficacy and highlight the therapeutic potential of IFNγ against alphaviruses. Lastly, in a collaborative effort to respond to the COVID-19 pandemic, we also explored and characterized the use of a new class of antiviral drugs. With the advent of increasing drug resistance, it is essential to develop novel and resilient therapeutics. We demonstrated the first antiviral potential of rhodium organometallics, which was previously shown to be effective against multiple antibiotic-resistant bacteria. Two complexes demonstrated high virucidal activity against SARS-CoV-2 and low toxicity in mammalian cell lines. Moreover, these complexes can be further derivatized to improve efficacy, making them a promising new antiviral strategy.

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.

Made available in DSpace on 2015-04-22T22:06:36Z (GMT). No. of bitstreams: 1 Dissertacao Final Gustavo Henrique.pdf: 11557113 bytes, checksum: 738eeceeb5f826798bc9ce2b09633f72 (MD5) Previous issue date: 2009-03-06 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Arbovirosis are currently recognized by the World Health Organization as a global problem. Most arbovirosis are summarized in diseases with acute and nonspecific symptoms such as fever, headache and muscle pain. Although self limited, these symptoms create relevant social and economic impacts. In Brazil, the arbovirosis caused by viruses belonging to the genus Alphavirus, Flavivirus and Orthobunyavirus and are the main cause of outbreaks or epidemics. This study aimed to study the circulation of arbovirus mayaro, venezuelan equine encephalitis virus, yellow fever virus, Saint Louis encephalitis virus and oropouche virus in a rural and an urban area of the State of Amazonas, Brazil. Therefore, the serology for detection of immunoglobulin G was used to assess the prevalence of antibodies against these viruses in 335 residents of a rural community in the state of Amazonas and PCR was used to assess the incidence of these viruses in 250 samples collected in urban area of Manaus. The results for serology suggest the movement of mayaro virus in the rural community. The seroprevalence detected in the samples was 41.5%. There was no significant relationship to risk for mayaro infection between genders (p value = 0.7760) or between age groups (p value = 0.9422). The positive serology detected among 39 children younger than 10 years indicates a recent infection. The factors of protection against mayaro infection detected were the use of mosquito net (p value = 0.0119) and the presence of animals in surrounding (p value = 0.0407). The risk factors identified for mayaro infection were the location of residence in towns near the forest (p value <0.0001) and presence of toilet in or near the home (p value = 0.0415). The serological results suggest that infection with mayaro occurred less than 10 years in the vicinity of residences analyzed. Molecular analysis of the samples collected in the urban area of Manaus not detected genomic fragments of arboviruses. Factors such as low viremia at the time of blood collection and storage of serum samples may have contributed to the non-detection of genomic fragments. However, the protocol for the detection of genomic fragments of arboviruses based on the PCR technique is already used in research centers and surveillance of Fundação de Medicina Tropical do Amazonas FMTAM and Instituto Leônidas e Maria Deane ILMD/FIOCRUZ. / As arboviroses são atualmente reconhecidas pela Organização Mundial da Saúde como um problema global. A maioria das arboviroses resume-se em afecções com sintomatologias agudas e inespecíficas, como febre, dores de cabeça e dores musculares. Embora sejam auto limitados, tais sintomas geram impactos sociais e econômicos relevantes. No Brasil, as arboviroses provocadas pelos por vírus pertencentes aos gêneros Alphavirus, Orthobunyavirus e Flavivirus são as principais causadoras de surtos ou epidemias. O presente estudo teve como objetivo estudar a circulação dos arbovírus mayaro, vírus da encefalite eqüina venezuelana, vírus da febre amarela, vírus da encefalite de Saint Louis e vírus oropouche em uma área rural e uma área urbana do Estado do Amazonas, Brasil. Assim sendo, a sorologia para detecção de imunoglobulinas G foi utilizada para avaliar a prevalência de anticorpos contra tais vírus em 335 moradores de uma comunidade rural do Estado do Amazonas e a PCR foi utilizada para avaliar a incidência de tais vírus em 250 amostras coletadas na área urbana de Manaus. Os resultados encontrados para a sorologia sugerem a circulação do vírus mayaro na comunidade rural. A soroprevalência detectada nas amostras foi de 41,5%. Não houve relação estatisticamente significativa de risco para a infecção por mayaro entre os gêneros (p valor=0,7760) ou entre as faixas etárias (p valor=0,9422). A sorologia positiva detectada entre 39 crianças menores de 10 anos indica uma infecção recente. Os fatores de proteção contra a infecção por mayaro detectados foram o uso de mosquiteiro (p valor=0,0119) e a presença de animais no peridomicílio (p valor=0,0407). Os fatores de risco detectados para a infecção por mayaro foram a localização do domicílio em vilas próximas à floresta (p valor<0,0001) e a presença de toalete dentro ou próximo ao domicílio (p valor=0,0415). Os resultados sorológicos sugerem que a infecção por mayaro ocorreu há menos de 10 anos nas proximidades das residências analisadas. A análise molecular das amostras coletadas na zona urbana de Manaus não detectou fragmentos genômicos de arbovírus. Fatores como baixa viremia no momento da coleta de sangue e estocagem das amostras de soro podem ter contribuído para a não detecção dos fragmentos genômicos. Contudo, o protocolo de detecção de fragmentos genômicos de arbovírus baseado na técnica de PCR está em uso nos centros de pesquisa e vigilância epidemiológica da Fundação de Medicina Tropical do Amazonas FMTAM e do Instituto Leônidas e Maria Deane ILMD/FIOCRUZ.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-21T14:53:07Z No. of bitstreams: 1 DISS_2014_Nayara Zuchi.pdf: 3111436 bytes, checksum: 8b58de352642d734fc2fdd8891b32e5e (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-26T12:55:13Z (GMT) No. of bitstreams: 1 DISS_2014_Nayara Zuchi.pdf: 3111436 bytes, checksum: 8b58de352642d734fc2fdd8891b32e5e (MD5) / Made available in DSpace on 2017-09-26T12:55:13Z (GMT). No. of bitstreams: 1 DISS_2014_Nayara Zuchi.pdf: 3111436 bytes, checksum: 8b58de352642d734fc2fdd8891b32e5e (MD5) Previous issue date: 2014-03-27 / CAPES / Introdução: O gênero Alphavirus, família Togaviridae, alberga arbovírus de importância médica relatados em áreas tropicais mundialmente. Nas Américas, os alfavírus de maior importância compreendem os das encefalites equinas e o vírus Mayaro (MAYV). No Brasil, o MAYV tem sido relatado em epidemias de doença febril principalmente no norte do país. O principal objetivo deste estudo foi investigar a situação epidemiológica de alfavírus em pacientes febris durante epidemia de dengue em Mato Grosso (MT). Material e Métodos: Entre 2011 e 2012, 604 amostras de soro de pacientes com doença febril aguda suspeita de dengue durante epidemia em MT foram submetidas a Duplex-RT-PCR seguida de Multiplex-semi-nested-PCR para pesquisa dos alfavírus MAYV, vírus Aura e os vírus das encefalites equinas do Leste, Oeste e Venezuelana. Amostras positivas foram confirmadas em dois testes independentes e os produtos de PCR submetidos a sequenciamento nucleotídico. Amostras positivas foram submetidas a RT-PCR em tempo real (RT-qPCR) e isolamento viral em cultura de células. Todas as amostras foram também investigadas para flavivirus em um estudo paralelo. Resultados: Foram encontrados 15/604 (2,5 %) pacientes positivos para o MAYV em Cuiabá (9), Várzea Grande (3), Nossa Senhora do Livramento (1) e Sorriso (2). Destes, 12 (80,0 %) apresentaram co-infecções com DENV-4 e 3 (20,0 %) infecções únicas pelo MAYV. Dentre 13 amostras submetidas a RT-qPCR, 10 (76,9 %) apresentaram carga viral entre log 0,965-3,321 cópias/μL. Discussão: Casos esporádicos de infecção pelo MAYV foram identificados durante uma grande epidemia de dengue no MT em residentes de áreas urbanas, sem histórico recente de viagem ou visita a áreas rurais e/ou silvestres. A ocorrência do MAYV em estados adjacentes, em cidades afetadas pela rodovia Cuiabá-Santarém e soroprevalência em índios Xavantes no estado corroboram a evidência da circulação de MAYV no MT. Apesar do MAYV ser transmitido principalmente por Haemagogus janthinomys em áreas silvestres, as evidências encontradas no presente estudo sugerem a circulação de MAYV em área urbana de MT. Contudo, o ciclo de transmissão do vírus no estado não foi elucidado. A evidência de circulação do MAYV em indivíduos febris durante epidemia de dengue em área urbana deve ser motivo de atenção das autoridades locais de saúde pública para a eventual circulação silenciosa de outros arbovírus no estado. / Introduction: The Alphavirus genus, Togaviridae family, comprises arboviruses of medical importance reported in tropical areas worldwide. In the Americas, the most important alfaviruses are the equine encephalitis group and Mayaro virus (MAYV). In Brazil, MAYV has been reported in outbreaks of febrile illness mainly in the North region of the country. The aim of this study was to investigate the epidemiological situation of alfaviruses in febrile patients during a dengue outbreak in Mato Grosso (MT). Material and methods: Between 2011 and 2012 in MT, 604 serum samples collected from patients suspected of acute febrile illness were submitted to Duplex-RT-PCR followed by Multiplex-semi-nested-PCR for MAYV, Aura virus and East, West and Venezuelan equine encephalitis viruses. Positive samples were confirmed twice in independent tests and, PCR products were submitted to nucleotide sequencing. Positive samples were also submitted to Real time RT-PCR (RT-qPCR) and inoculation in cell culture. The samples were also investigated for flaviviruses in a parallel study. Amostras positivas foram submetidas a RT-PCR em tempo real (RT-qPCR) e isolamento viral em cultura de células. Todas as amostras foram também investigadas para flavivirus em um estudo paralelo. Results: 15/604 (2.5 %) patients from Cuiabá (9), Várzea Grande (3), Nossa Senhora do Livramento (1) and Sorriso (2) were positive for MAYV; 12 (80 %) are co-infected with DENV-4 and 3 (20 %) are single infections with MAYV. Co-infected patients presented a wider variety of clinical manifestations. Among 13 samples tested by RT-qPCR, 10 (76.9 %) presented viral load ranging from log 0,965-3,321 copies/μL. Discussion: Sporadic infections with MAYV were identified during a massive Dengue outbreak in MT in residents of urban areas without recent history of travel or visit to rural or sylvatic areas. The occurrence of MAYV infections in neighboring states, including cities affected by the Cuiabá-Santarém highway and seroprevalence in Xavante Indians from MT, corroborate the evidence of MAYV circulation in MT. Despite MAYV is transmitted mainly by Haemagogus janthinomys in sylvatic areas, the evidence found in this study suggests the circulation of MAYV in urban areas of MT. However, the transmission cycle of MAYV in MT remains to be determined. The evidence of MAYV circulation in febrile individuals during a dengue outbreak in urban areas should cause concerns in the local public health authorities about the eventual silent circulation of arboviruses in the state.

Arthropod-borne-viruses (arboviruses) pose a global threat due to their ability to be transmitted by hematophagous insects to vertebrate hosts resulting in a range of serious infectious diseases. Sindbis virus (SINV) is the prototype arbovirus of the genus Alphavirus in the family Togaviridae. The purpose of this study was to investigate the use of a fluorescent tagged reporter virus in both in vitro and in vivo environments. The fluorescent protein GFP was inserted between the Capsid and PE2 in the genome of TR339; SINV TaV-GFP (Wm. Klimstra Lab). This virus construct should have the same infectivity and virulence as wild type TR339, leaving a fluorescent ‘path’ in infected cells that may reveal virus transit. Virus stocks were grown in BHK-21 vertebrate cells and C7-10 mosquito cells. Two Aedes albopictus mosquito cell lines, C7-10 and C6/36, were then challenged with vertebrate and mosquito grown reporter virus. Evidence of GFP were seen as early as 6 hours post infection (p.i.) in all samples. Infected C7-10 cells with the vertebrate grown reporter virus were fixed for 1 hour in chilled 4% buffered paraformaldehyde; GFP was shown to be resilient to both fixation and light quenching. Ultimately, Ae. aegypti mosquitoes were challenged with a viremic bloodmeal at a titer of 107 PFU/ml and midguts were dissected over several days. The presence of GFP was observed in midgut columnar epithelial cells as early as day 3 p.i. and remained localized even at day 30 p.i. This is in agreement with published work on the interaction of TR339 in Ae. aegypti gut, signaling this viral construct as a means to visualize wild-type infection.