Introduction: Dengue (DENV) and Zika (ZIKV) viruses are flaviviruses that infect millions of people each year causing epidemics, which can cause mild to severe consequences in infected patients and can even lead to death. However, to date there are no effective ways to prevent or treat infections caused by these viruses. Thus, the development of antiviral drugs for DENV and ZIKV is urgent. Objective: The aims is identify drug candidates against DENV and ZIKV NS3 helicase and NS2B-NS3 protease proteins, which are essential in the viral life cycle, using an integration of computational and experimental strategies. Methods: This dissertation was divided in two projects. In the first project a virtual screening (VS) based on molecular docking and machine learning models (ML) was used to prioritize compounds that inhibit DENV helicase and protease proteins. To do this, binary ML models were developed and validated to select potential compounds with activity against DENV proteins. In the virtual screening of compounds against DENV protease, in addition to docking and ML models, models based on the molecular shape and volume (shape-based) generated and validated from DENV NS2B-NS3 protease inhibitors identified in the literature were also used. In the second project, we used a molecular docking approach and ML models to prioritize compounds against ZIKV NS2B-NS3 protease and NS3 helicase. Results: At the virtual screening performed in project 1, it was possible to prioritize 19 compounds, 9 compounds for NS3 helicase and 10 for NS2B-NS3 protease. These compounds have already been purchased and are in the experimental evaluation phase. In project 2, 22 compounds were selected, 15 for NS3 helicase and 7 for NS2B-NS3 protease. Phenotypic assays in ZIKV infected cells, showed that 6 compounds demonstrated inhibitory activity against the virus and are being validated in enzymatic assays. Conclusion: the present work demonstrated the potential of integration of computational techniques and experimental assays to accelerate the identification of potential candidates for Dengue and Zika virus antiviral candidates.

Dengue and zika are viral infectious diseases occurring in countries with a tropical and subtropical climate in which around 3.6 billion people live. It is estimated that in 50 to 100 million new cases of dengue occur annually, generating economic, social and public health impacts. Dengue and zika have usually been diagnosed by serological methods which are generally of low confidence, as they can generate false-positive results, which are related to the presence of antibodies produced against previous infections of the virus. The molecular methods are more accurate, however the molecular method most commonly used (PCR) requires a long time of reaction and requires sophisticated instrumentation and high cost, making point of care applications difficult,, especially in developing countries. This work presents the development of a molecular diagnostic methodology in a paper-based platform that allowed the detection of the virus through the reverse transcription -loop mediated isothermal amplification (RT-LAMP). The reactions were carried out on 6 mm diameter FTA paper discs, confined in a multi-layered polyester-toner device, incubated at 65 ° C for 45 minutes in a dry bath and then performed visual detection using the SYBR Green intercalator. Positive reactions were identified by the green fluorescence emitted after the addition of the intercalator. The results were recorded through the capture of the images by a photodocumentator and/or by smartphone and later analyzed by the software ImageJ, allowing the comparison between negative and positive reactions. The methodology developed for the detection of the virus by RT-LAMP in paper substrate was sensitive, being able to detect the virus in initial concentrations of 0.1 pg μL -1 of RNA in the master mixture. In addition, it was possible to detect the virus directly in complex samples (serum of infected patients) without the need of previous viral RNA extraction step. Elimination of the RNA extraction step together with the visual detection on the paper produce the final result in 46 minutes. The results demonstrated that the detection of the virus by RT-LAMP in paper substrates is a valuable tool for the molecular diagnosis of infectious diseases, presenting great potential for point-of-care applications for both diagnostics and epidemiological studies, especially in developing countries.

Flavivirus-induced pathogens are considered a serious public health problem in the world, because they affect primarily the poor populations of underdeveloped tropical countries. This family of viruses has an enveloped positive RNA genome encoding tree structural proteins and seven non-structural proteins. In particular, non-structural protein 1 (NS1) is associated mainly with the viral replication process and immune system leakage, and can be found in different glycosylated oligomeric forms, but only "mature" after its dimerization, or when in the hexamer form. In this context, inhibition of the dimerization process of NS1 has been pointed as a great target for the rational design of drugs. However, for this type of rationalized approach to be successful, it is necessary to understand the molecular mechanisms involved in the process of dimerization, as well as characterize its functional regions. In this study, through Molecular Dynamics (DM) simulations, the structural behavior of the NS1's of the Dengue and Zika were evaluated and compared in the monomeric and dimeric forms, from structures crystallized with glycosylations. For the structure of the Dengue (PDB: 5k6k), there was a need to previously model the regions of missing residues, then making necessary to replace the sugars in the structures. All of the N-glycosylations and intercysteine bonds of each monomer and dimer were performed in the Amber14 software. Specific histidine protonation was predicted by the H++ assay before proceeding with DM. All structures were submitted to DM by 100ns using the GROMACS 5.1.2 software. The stability and flexibility of the structures observed in the trajectories were used to elucidate the mechanisms responsible for the monomer-monomer interaction, as well as to understand the singularities between the regions that stood out. In the monomeric form of the two species, large conformational fluctuations were observed in their N-terminal loops involved in the β-roll composition, which in the dimeric form were stabilized by the triad Ile/Val19 - Phe20 - Ile21 for creating a framework favorable to the interlacing of the N-terminal loops between the A and B chains. Concomitantly, Lysines-189 of the β-ladder domain acted as a key residue (staple) to stabilize the central portion of β-roll. The stability mechanisms of NS1 revealed in this work for the Zika and Dengue species, contribute to make the possibility of pharmacological intervention more accessible, in a rationalized way, whose approach could be directed at specific structural points responsible for its biological role.

A febre da Zika é uma arbovirose, transmitida principalmente por mosquitos do gênero Aedes, mas há também a possibilidade de transmissão vertical, com relatos alarmantes de casos de Síndrome Congênita da Zika associadas à infecção. Sabe-se que o vírus da Zika (ZIKV) atravessa a placenta e infecta o feto, mas a patogênese dessa doença ainda não foi bem esclarecida. A partir disso, o presente estudo visou compreender melhor as consequências que a infecção pelo ZIKV causa no tecido placentário humano e nos tecidos de um natimorto, assim como nas linhagens de células de mastócitos HMC-1 e trofoblásticas HTR-8/SVneo. Os tecidos foram submetidos à análise histopatológica, imunoensaios e hibridização in situ para detecção viral, de células imunes e citocinas/mediadores inflamatórios, além da avaliação ultraestrutural das células da placenta. Foram realizados os ensaios de PRNT e RT-PCR tempo real no soro das pacientes. Nas linhagens de células, foi padronizado um protocolo de infecção após ensaio de cinética in vitro utilizando imunofluorescência e/ou citometria de fluxo. Em seguida, analisamos as alterações ultraestruturais das células infectadas por microscopia eletrônica de transmissão, observamos a degranulação e liberação de citocinas nos mastócitos ou o estresse oxidativo nas células placentárias. Danos histológicos foram encontrados nas placentas, como a imaturidade vilosa, deposição de fibrina, infiltrado inflamatório, áreas de calcificação e diminuição dos componentes de matriz extracelular. Detectamos células positivas para os antígenos de ZIKV, comprovando inclusive a replicação com a marcação da proteína NS1 e do RNA negativo em diferentes células placentárias e células imunes residentes ou circulantes. O aumento da celularidade (células CD68+ e linfócitos TCD8+) e da expressão de citocinas pró-inflamatórias locais como IFN-ɣ e TNF-α e outros mediadores, como RANTES/CCL5, MMPs e VEGFR-2, sustentam o caráter da inflamação e disfunção placentária causadas pelo vírus. Além disso, notamos que a diminuição da neurotrofina BDNF pode modular os danos neuronais fetais, já que sua expressão foi menor nas placentas de pacientes que tiveram bebês com microcefalia. Foram observadas também alterações histopatológicas, detecção do vírus e evidências de replicação em todos os órgãos do natimorto que foram analisados. Os aspectos ultraestruturais das células infectadas tanto nos tecidos quanto nas culturas de células apresentaram modificações nas organelas, principalmente no retículo endoplasmático e nas mitocôndrias. Observamos que os mastócitos são permissivos à infecção tanto no tecido placentário quanto in vitro e a infecção estimula a degranulação e liberação de citocinas e mediadores. As células trofoblásticas infectadas apresentaram aumento significativo da atividade da enzima antioxidante SOD, das espécies reativas MDA e do ON, enquanto a atividade enzimática de CAT foi menor nas células infectadas pelo ZIKV. Com nossos achados, podemos afirmar que as alterações placentárias causadas pelo ZIKV não são patognomônicas, no entanto, esta infecção leva a alterações placentárias graves, e a infecção algumas vezes resulta no comprometimento ou dano fetal. Os achados apresentados neste trabalho ajudam a entender a imunopatogênese da Zika no contexto materno-fetal.

Qualitative research to elucidate social constructions about dengue, chikungunya and zika in an endemic municipality of Colombia. Sixty-one people with subjective experience of disease participated. Semistructured and open interviews, informal dialogues and observation notes record were used. The analysis was performed following the inductive method of social research. Two ways of understanding dengue are evident: as a “normal” disease or as a serious disease that can cause death. Chikungunya and zika are conceived as new diseases, difficult to recognize, that fall into social oblivion due to the low occurrence of cases. Two therapeutic itineraries for the three diseases are identified, mediated by the severity of the symptoms and the perception of the attention received by the health services. Drugstores are configured as a key element of therapeutic itineraries. It is concluded that the social construction of these diseases is crossed by a tension between recognition and forgetfulness. Public policies and actions aimed at the prevention and control of these events have the challenge of facing the “forgetfulness” that daily life brings, therefore they should not be limited to approaches that ignore the ways in which diseases are lived and faced.

A pesquisa avalia se o monitoramento de mídias sociais pode ser uma ferramenta de previsão de padrões epidemiológicos. Para tanto, foram monitoradas as mídias sociais Facebook, Twitter, Instagram, Flickr, Youtube e blogs no Estado de Santa Catarina entre 24 de janeiro de 2016 e 27 de fevereiro de 2016. Os rumores sobre dengue, chikungunya, zika, Aedes aegypti e microcefalia foram relacionados ao número de casos suspeitos, confirmados ou descartados pela Vigilância Epidemiológica de Santa Catarina no mesmo período. Os resultados obtidos mostram que há forte correlação entre as variáveis e que o monitoramento pode ser usado como um modelo preditivo por profissionais da saúde e gestores públicos.

Few Zika virus (ZIKV) outbreaks had been reported since its first detection in 1947, until the recent epidemics occurred in South America (2014/2015) and expeditiously became a global public health emergency. This arbovirus reached 0.5-1.3 million cases of ZIKV infection in Brazil in 2015 and rapidly spread in new geographic areas such as the Americas. Despite the mild symptoms of the Zika fever, the major concern is related to the related severe neurological disorders, especially microcephaly in newborns. Advances in ZIKV drug discovery have been made recently and constitute promising approaches to ZIKV treatment. In this review, we summarize current computational drug discovery efforts and their applicability to discovery of anti-ZIKV drugs. Lastly, we present successful examples of the use of computational approaches to ZIKV drug discovery.

OBJECTIVE: To analyze the spatial distribution and identify high-risk spatial clusters of Zika, dengue, and chikungunya (ZDC), in the city of Rio de Janeiro, Brazil, and their socioeconomic status. METHODS: An ecological study based on data from a seroprevalence survey. Using a rapid diagnostic test to detect the arboviruses, 2,114 individuals were tested in 2018. The spatial distribution was analyzed using kernel estimation. To detect high-risk spatial clusters of arboviruses, we used multivariate scan statistics. The Social Development Index (SDI) was considered in the analysis of socioeconomic status. RESULTS: Among the 2,114 individuals, 1,714 (81.1%) were positive for at least one arbovirus investigated. The kernel estimation showed positive individuals for at least one arbovirus in all regions of the city, with hot spots in the North, coincident with regions with very low or low SDI. The scan statistic detected three significant (p<0.05) high-risk spatial clusters for Zika, dengue, and chikungunya viruses. These clusters correspond to 35.7% (n=613) of all positive individuals of the sample. The most likely cluster was in the North (cluster 1) and overlapped regions with very low and low SDI. Clusters 2 and 3 were in the West and overlapping regions with low and very low SDI, respectively. The highest values of relative risks were in cluster 1 for CHIKV (1.97), in cluster 2 for ZIKV (1.58), and in cluster 3 for CHIKV (1.44). Regarding outcomes in the clusters, the Flavivirus had the highest frequency in clusters 1, 2, and 3 (42.83%, 54.46%, and 52.08%, respectively). CONCLUSION: We found an over-risk for arboviruses in areas with the worst socioeconomic conditions in Rio de Janeiro. Moreover, the highest concentration of people negative for arboviruses occurred in areas considered to have better living conditions.

Dengue virus (DENV), which can lead to fatal hemorrhagic fever, affects 390 million people worldwide. The closely related Zika virus (ZIKV) causes microcephaly in newborns and Guillain-Barré syndrome in adults. Both viruses are mostly transmitted by Aedes albopictus and Aedes aegypti mosquitoes, which, due to globalization of trade and travel alongside climate change, are spreading worldwide, paving the way to DENV and ZIKV transmission and the occurrence of new epidemics. Local outbreaks have already occurred in temperate climates, even in Europe. As there are no specific treatments, these viruses are an international public health concern. Here, we analyze and discuss DENV and ZIKV outbreaks history, clinical and pathogenesis features, and modes of transmission, supplementing with information on advances on potential therapies and restraining measures. Taking advantage of the knowledge of the structure and biological function of the capsid (C) protein, a relatively conserved protein among flaviviruses, within a genus that includes DENV and ZIKV, we designed and patented a new drug lead, pep14-23 (WO2008/028939A1). It was demonstrated that it inhibits the interaction of DENV C protein with the host lipid system, a process essential for viral replication. Such an approach can be used to develop new therapies for related viruses, such as ZIKV.