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Recurso educacional aberto em Pt-br | CVSP - Brasil | ID: cvsp-brasil--299459
ResumoInfográfico elaborado por Cristiane Albuquerque e Vinícius Ferreira do Instituto Oswaldo Cruz/Fiocruz que descrevem a etapa inicial do projeto 'Eliminar a Dengue: desafio Brasil'. Luciano Moreira, pesquisador da Fiocruz é o coordenador do projeto. Já realizada com sucesso na Austrália, Vietnã e Indonésia, a fase de estudos de campo conta com a liberação de mosquitos Aedes aegypti com a bactéria Wolbachia. O projeto tem o apoio do Instituto Oswaldo Cruz (IOC/Fiocruz), do Centro de Pesquisas René Rachou (Fiocruz/Minas) e do Programa de Computação Científica (PROCC/Fiocruz). O primeiro local a participar é o bairro de Tubiacanga, na Ilha do Governador, na cidade do Rio de Janeiro, estudado pela equipe do projeto desde 2012. Esta é a primeira vez que um país nas Américas recebe o estudo.
Fiocruz inicia estudo com mosquitos que podem reduzir a transmissão da dengue no IOC Fiocruz (vídeo)
Recurso educacional aberto em Pt-br | CVSP - Brasil | ID: cvsp-brasil--299560
ResumoVídeo da etapa inicial do Projeto ‘Eliminar a Dengue: Desafio Brasil’ com o coordenador do projeto, o pesquisador Luciano Moreira que explica os estudos já realizados com sucesso na Austrália, Vietnã e Indonésia, a fase de estudos de campo conta com a liberação de mosquitos Aedes aegypti com a bactéria Wolbachia que objetiva reduzir a transmissão do vírus do mosquito da dengue. O primeiro local a participar é o bairro de Tubiacanga, localizado na Ilha do Governador, na cidade do Rio de Janeiro, e estudado pela equipe do projeto desde 2012. Esta é a primeira vez em que um país nas Américas recebe o estudo. O projeto conta com a participação do Instituto Oswaldo Cruz (IOC/Fiocruz), Centro de Pesquisas René Rachou (Fiocruz/Minas) e Programa de Computação Científica (PROCC/Fiocruz).
Societal impact of dengue outbreaks: Stakeholder perceptions and related implications. A qualitative study in Brazil, 2015.
PLoS Negl Trop Dis; 11(3): e0005366, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28278157
ResumoBACKGROUND: The growing burden of dengue in many countries worldwide and the difficulty of preventing outbreaks have increased the urgency to identify alternative public health management strategies and effective approaches to control and prevent dengue outbreaks. The objectives of this study were to understand the impact of dengue outbreak on different stakeholders in Brazil, to explore their perceptions of approaches used by governmental authorities to control and prevent dengue outbreaks and to define the challenges and implications of preventing future outbreaks. METHODS: In 2015, a qualitative study was conducted in two urban states in Brazil: São Paulo, which was experiencing an outbreak in 2015, and Rio de Janeiro, which experienced outbreaks in 2011 and 2012. Face-to-face interviews using a semi-structured questionnaire were conducted with nine different categories of stakeholders: health workers (physicians, nurses), hospital administrators, municipal government representatives, community members and leaders, school administrators, business leaders and vector control managers. Interviews were focused on the following areas: impact of the dengue outbreak, perceptions of control measures implemented by governmental authorities during outbreaks and challenges in preventing future dengue outbreaks. RESULTS: A total of 40 stakeholders were included in the study. Health workers and community members reported longer waiting times at hospitals due to the increased number of patients receiving care for dengue-related symptoms. Health workers and hospital administrators reported that there were no major interruptions in access to care. Overall financial impact of dengue outbreaks on households was greatest for low-income families. Despite prevention and control campaigns implemented between outbreak periods, various stakeholders reported that dengue prevention and control efforts performed by municipal authorities remained insufficient, suggesting that efforts should be reinforced and better coordinated by governmental authorities, particularly during outbreak periods. CONCLUSION: The study shows that a dengue outbreak has a multisectorial impact in the medical, societal, economic and political sectors. The study provides useful insights and knowledge in different stakeholder populations that could guide local authorities and government officials in planning, designing and initiating public health programs. Research focused on a better understanding of how communities and political authorities respond to dengue outbreaks is a necessary component for designing and implementing plans to decrease the incidence and impact of dengue outbreaks in Brazil.
Principal Components Analysis Based Unsupervised Feature Extraction Applied to Gene Expression Analysis of Blood from Dengue Haemorrhagic Fever Patients.
Sci Rep; 7: 44016, 2017 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-28276456
ResumoDengue haemorrhagic fever (DHF) sometimes occurs after recovery from the disease caused by Dengue virus (DENV), and is often fatal. However, the mechanism of DHF has not been determined, possibly because no suitable methodologies are available to analyse this disease. Therefore, more innovative methods are required to analyse the gene expression profiles of DENV-infected patients. Principal components analysis (PCA)-based unsupervised feature extraction (FE) was applied to the gene expression profiles of DENV-infected patients, and an integrated analysis of two independent data sets identified 46 genes as critical for DHF progression. PCA using only these 46 genes rendered the two data sets highly consistent. The application of PCA to the 46 genes of an independent third data set successfully predicted the progression of DHF. A fourth in vitro data set confirmed the identification of the 46 genes. These 46 genes included interferon- and heme-biosynthesis-related genes. The former are enriched in binding sites for STAT1, STAT2, and IRF1, which are associated with DHF-promoting antibody-dependent enhancement, whereas the latter are considered to be related to the dysfunction of spliceosomes, which may mediate haemorrhage. These results are outcomes that other type of bioinformatic analysis could hardly achieve.
BMC Infect Dis; 17(1): 204, 2017 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-28284209
ResumoBACKGROUND: Dengue is an acute febrile illness considered the major arboviral disease in terms of morbidity, mortality, economic impact and dissemination worldwide. Brazil accounts for the highest notification rate, with circulation of all four dengue serotypes. The NS1 antigen is a dengue highly conserved specific soluble glycoprotein essential for viral replication and viability that can be detected 0 to 18 days from the onset of fever (peak first 3 days). It induces a strong humoral response and is known as a complement-fixing antigen. Lower NS1 test sensitivity occurs in secondary dengue infections probably due to immune complex formation impairing antigen detection by ELISA. METHODS: We compared the sensitivity of NS1 ELISA in heat dissociated and non-dissociated samples from 156 RT-PCR confirmed acute dengue-4 cases from 362 prospectively enrolled patients. RESULTS: Secondary infections accounted for 83.3% of cases. NS1 ELISA was positive in 42.5% and indeterminate in 10.2% of dengue-4 cases. After heat dissociation, 7 negative and 16 indeterminate samples turned positive, increasing the overall test sensitivity to 57.7%. CONCLUSIONS: Although it is time consuming and requires the use of specific laboratory equipment, NS1 ELISA combined with heat dissociation could be a slightly better alternative for triage in suspected dengue cases.
Mathematical modelling and a systems science approach to describe the role of cytokines in the evolution of severe dengue.
BMC Syst Biol; 11(1): 34, 2017 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-28284213
ResumoBACKGROUND: Dengue causes considerable morbidity and mortality in Sri Lanka. Inflammatory mediators such as cytokines, contribute to its evolution from an asymptotic infection to severe forms of dengue. The majority of previous studies have analysed the association of individual cytokines with clinical disease severity. In contrast, we view evolution to Dengue Haemorrhagic Fever as the behaviour of a complex dynamic system. We therefore, analyse the combined effect of multiple cytokines that interact dynamically with each other in order to generate a mathematical model to predict occurrence of Dengue Haemorrhagic Fever. We expect this to have predictive value in detecting severe cases and improve outcomes. Platelet activating factor (PAF), Sphingosine 1- Phosphate (S1P), IL-1ß, TNFα and IL-10 are used as the parameters for the model. Hierarchical clustering is used to detect factors that correlated with each other. Their interactions are mapped using Fuzzy Logic mechanisms with the combination of modified Hamacher and OWA operators. Trapezoidal membership functions are developed for each of the cytokine parameters and the degree of unfavourability to attain Dengue Haemorrhagic Fever is measured. RESULTS: The accuracy of this model in predicting severity level of dengue is 71.43% at 96 h from the onset of illness, 85.00% at 108 h and 76.92% at 120 h. A region of ambiguity is detected in the model for the value range 0.36 to 0.51. Sensitivity analysis indicates that this is a robust mathematical model. CONCLUSIONS: The results show a robust mathematical model that explains the evolution from dengue to its serious forms in individual patients with high accuracy. However, this model would have to be further improved by including additional parameters and should be validated on other data sets.
Parasit Vectors; 10(1): 140, 2017 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-28284225
ResumoBACKGROUND: Global warming has a marked influence on the life cycle of epidemic vectors as well as their interactions with human beings. The Aedes albopictus mosquito as the vector of dengue fever surged exponentially in the last decade, raising ecological and epistemological concerns of how climate change altered its growth rate and population dynamics. As the global warming pattern is considerably uneven across four seasons, with a confirmed stronger effect in winter, an emerging need arises as to exploring how the seasonal warming effects influence the annual development of Ae. albopictus. METHODS: The model consolidates a 35-year climate dataset and designs fifteen warming patterns that increase the temperature of selected seasons. Based on a recently developed mechanistic population model of Ae. albopictus, the model simulates the thermal reaction of blood-fed adults by systematically increasing the temperature from 0.5 to 5 °C at an interval of 0.5 °C in each warming pattern. RESULTS: The results show the warming effects are different across seasons. The warming effects in spring and winter facilitate the development of the species by shortening the diapause period. The warming effect in summer is primarily negative by inhibiting mosquito development. The warming effect in autumn is considerably mixed. However, these warming effects cannot carry over to the following year, possibly due to the fact that under the extreme weather in winter the mosquito fully ceases from development and survives in terms of diapause eggs. CONCLUSIONS: As the historical pattern of global warming manifests seasonal fluctuations, this study provides corroborating and previously ignored evidence of how such seasonality affects the mosquito development. Understanding this short-term temperature-driven mechanism as one chain of the transmission events is critical to refining the thermal reaction norms of the epidemic vector under global warming as well as developing effective mosquito prevention and control strategies.
Virus Res; 232: 139-151, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-28267608
ResumoDengue virus (DENV) is the most important arbovirus in the world; DENV is transmitted by the Aedes genus of mosquitoes and can establish a life-long persistent infection in mosquitoes. However, the exact mechanism by which persistent infection is established remains unknown. In this study the differential expression of miRNAs was analysed by deep sequencing and RT-qPCR using a previously established C6/36-HT cell line persistently infected with DENV 2 (C6-L) as a model. miR-927, miR-87, miR-210, miR-2a-3p, miR-190 and miR-970 were up-regulated, whereas miR-252, miR-263a-3p, miR-92b, miR-10-5p miR-9a-5p, miR-9a-1, miR-124, miR-286a and miR-286b were down-regulated in C6-L cells compared with C6/36 cells acutely infected with the same virus or mock-infected cells. Deep sequencing results were validated by RT-qPCR for the highly differentially expressed miR-927 and miR-9a-5p, which were up- and down-regulated, respectively, compared with both acutely and mock-infected C6/36 cells. The putative targets of these miRNAs include components of the ubiquitin conjugation pathway, vesicle-mediated transport, autophagy, and the JAK-STAT cascade as well as proteins with endopeptidase activity. Other putative targets include members of the Toll signalling pathway and proteins with kinase, ATPase, protease, scavenger receptor or Lectin C-type activity or that participate in fatty acid biosynthesis or oxidative stress. Our results suggest that several specific miRNAs help regulate the cellular functions that maintain equilibrium between viral replication and the antiviral response during persistent infection of mosquito cells. This study is the first report of a global miRNA profile in a mosquito cell line persistently infected with DENV.
Longitudinal study of Thai people media exposure, knowledge, and behavior on dengue fever prevention and control.
J Infect Public Health; 2017 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-28285970
ResumoDengue hemorrhagic fever is transmitted through a bite by a dengue -infected Aedes aegypti mosquito. It was first reported in the mid -20th century in Thailand, and since then its epidemiology has been of great concern and has spread all across the country. The alarming incidence of dengue posed a serious threat to human health in all major cities of Thailand. This study was aimed at identifying the level of awareness of dengue fever in Thai population knowledge for prevention and control, and most importantly contribution of media in educating masses for dengue control measures. It is longitudinal in nature and was conducted in 25 provinces of Thailand during 2013-2015. Approximately 7772 respondents participated in this study, with the selection of provinces based on considerations like population, prevalence and demography. A pre-tested structured questionnaire was used to collect information relevant to study participants' demographic profile, pre-existing knowledge about dengue fever and its reinforcement through media, and population attitudes toward prevention and control. Over the period of three years, a positive trend was revealed relevant to the contribution of media in educating and reminding the Thai population of dengue, without any uniformity or powerful campaigns. Based on the results drawn from this study, we conclude that despite the measures undertaken to prevent dengue fever, there is insufficient media exposure. An interdisciplinary approach involving the community participation, media, and government is needed to overcome dengue threat in Thailand.
Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8 T cells.
Nat Microbiol; 2: 17036, 2017 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-28288094
ResumoCD8 T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-Î³ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/ß receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-Î³-ELISPOT and IFN-Î³-ICS on CD8 T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8 T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8 T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8 T cell responses that reduced infectious ZIKV levels, and CD8 T cell depletions confirmed that CD8 T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8 T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8 T cell responses of human relevance.
Preparation of MS2-based nanoparticles as control and standard materials for the molecular detection of dengue virus serotypes.
Virus Res; 2017 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-28288812
ResumoTo quantify dengue virus (DENV) and evaluate the performance of clinical laboratories using quantitative real-time polymerase chain reaction (qRT-PCR) assays, we constructed high-efficiency expression systems to produce DENV-1 to 4 nanoparticles and assessed their suitability as standard and control materials in 20 laboratories across China. Targeted gene sequences of DENV-1 to 4 were synthesized and inserted into pACYC-Duet 1-MS2 recombinant plasmids to generate corresponding nanoparticle expression systems. After collection, verification, and quantification by digital PCR (dPCR), DENV-1 to 4 nanoparticles were prepared as control and standard materials. Five positive and three negative samples of each DENV serotype in every panel were used for assessing the performance of the participating laboratories across China, as well as standard materials for the quantitative detection of DENV using qRT-PCR assays. The accuracy, sensitivity, and specificity of qRT-PCR used by the 20 evaluated laboratories were 89.6 (569/635), 85.1 (336/395), and 97.1% (233/240), respectively. Overall, sixteen (80.0%) laboratories were qualified in detecting DENV, among which five (25.0%) were designated as "competent", eleven (55.0%) were defined as "acceptable", and four (20%) were considered to be "improvable". The results generated from the DENV standard samples were significantly positively correlated with those generated by dPCR (r =0.8698, P<0.001). In summary, DENV nanoparticles could potentially be used as controls for improving the performance of laboratories and as standards for the quantitative detection of DENV.
Co-circulating serotypes in a dengue fever outbreak: Differential hematological profiles and phylogenetic relationships among viruses.
J Clin Virol; 90: 7-13, 2017 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-28292653
ResumoBACKGROUND: Dengue virus, represented by four distinct, genetically diverse serotypes, is the etiologic agent of asymptomatic to severe hemorrhagic diseases. The spatiotemporal dynamics of dengue serotypes and its association to specific diseases vary among the different regions worldwide. By 2007, and in São Paulo State, Brazil, dengue-case concentration in urban centers had changed to increased incidence in small- and medium-sized towns, the case of Marília. OBJECTIVES: The aim of this article was to distinguish dengue serotypes circulating during the 2007 Marília outbreak and define their association to demographic and hematological patient profiles, as well as the phylogenetic relationships among the different viruses. STUDY DESIGN: PCR amplicons corresponding to the junction of capsid and dengue pre-membrane encoding genes, obtained from dengue serologically positive patients, were sequenced. Hematological and demographic data of patients with different Dengue serotypes were evaluated by univariate and bivariate statistics. Dengue PCR sequences were used in phylogenetic relationships analyzed for maximum parsimony. RESULTS: Molecular typing confirmed co-circulation of the dengue serotypes 1 (DENV1) and 3 (DENV3), which presented divergent correlation patterns with regard to hematological descriptors. The increase in atypical lymphocytes, a likely indication of virus load, could be significantly associated to a decrease in leukocyte counts in the DENV3 group and platelet in the DENV1. Phylogenetic reconstitution revealed the introduction of DENV1 from northern Brazil and local divergence of DENV3 by either microevolution or viral introduction from other geographical regions or both. CONCLUSIONS: Dengue dynamics showed regional molecular-epidemiologic specificity, which has important implications for introduction of vaccines, disease management, and transmission control.
Knowledge and Practice Regarding Dengue and Chikungunya: a cross sectional study among Healthcare Workers and Community in Northern Tanzania.
Trop Med Int Health; 2017 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-28294472
ResumoOBJECTIVE: To investigate knowledge and prevention practices regarding dengue and chikungunya amongst community members, as well as knowledge, treatment and diagnostic practices among healthcare workers. METHOD: We conducted a cross-sectional survey with 125 community members and 125 healthcare workers from 13 health facilities in six villages in the Hai district of Tanzania. A knowledge score was generated based on participant responses to a structured questionnaire, with a score of 40 or higher (out of 80 and 50 total scores for community members and healthcare workers, respectively) indicating good knowledge. We conducted qualitative survey (n=40) to further assess knowledge and practice regarding dengue and chikungunya fever. RESULTS: 15.2% (n=19) of community members had good knowledge regarding dengue, whereas 53.6%, (n=67) of healthcare workers did. 20.3% (n=16) of participants from lowland areas and 6.5% (n=3) from highland areas had good knowledge of dengue (χ = 4.25, p=0.03). Only 2.4% (n=3) of all participants had a good knowledge score for chikungunya. In the qualitative study, community members expressed uncertainty about dengue and chikungunya. Some healthcare workers thought that they were new diseases. CONCLUSION: There is insufficient knowledge regarding dengue and chikungunya fever among community members and healthcare workers. Health promotion activities on these diseases based on Ecological Health Mode components to increase knowledge and improve preventive practices should be developed. This article is protected by copyright. All rights reserved.
Dengue Baidu Search Index data can improve the prediction of local dengue epidemic: A case study in Guangzhou, China.
PLoS Negl Trop Dis; 11(3): e0005354, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28263988
ResumoBACKGROUND: Dengue fever (DF) in Guangzhou, Guangdong province in China is an important public health issue. The problem was highlighted in 2014 by a large, unprecedented outbreak. In order to respond in a more timely manner and hence better control such potential outbreaks in the future, this study develops an early warning model that integrates internet-based query data into traditional surveillance data. METHODOLOGY AND PRINCIPAL FINDINGS: A Dengue Baidu Search Index (DBSI) was collected from the Baidu website for developing a predictive model of dengue fever in combination with meteorological and demographic factors. Generalized additive models (GAM) with or without DBSI were established. The generalized cross validation (GCV) score and deviance explained indexes, intraclass correlation coefficient (ICC) and root mean squared error (RMSE), were respectively applied to measure the fitness and the prediction capability of the models. Our results show that the DBSI with one-week lag has a positive linear relationship with the local DF occurrence, and the model with DBSI (ICC:0.94 and RMSE:59.86) has a better prediction capability than the model without DBSI (ICC:0.72 and RMSE:203.29). CONCLUSIONS: Our study suggests that a DSBI combined with traditional disease surveillance and meteorological data can improve the dengue early warning system in Guangzhou.
Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells.
PLoS Pathog; 13(3): e1006265, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28264033
ResumoThe Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.
Comput Math Methods Med; 2017: 2187390, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28293273
Resumo. Predicting the risk of severity at an early stage in an individual patient will be invaluable in preventing morbidity and mortality caused by dengue. We hypothesized that such predictions are possible by analyzing multiple parameters using mathematical modeling. . Data from 11 adult patients with dengue fever (DF) and 25 patients with dengue hemorrhagic fever (DHF) were analyzed. Multivariate statistical analysis was performed to study the characteristics and interactions of parameters using dengue NS1 antigen levels, dengue IgG antibody levels, platelet counts, and lymphocyte counts. Fuzzy logic fundamentals were used to map the risk of developing severe forms of dengue. The cumulative effects of the parameters were incorporated using the Hamacher and the OWA operators. . The operator classified the patients according to the severity level during the time period of 96 hours to 120 hours after the onset of fever. The accuracy ranged from 53% to 89%. . The results show a robust mathematical model that explains the evolution from dengue to its serious forms in individual patients. The model allows prediction of severe cases of dengue which could be useful for optimal management of patients during a dengue outbreak. Further analysis of the model may also deepen our understanding of the pathways towards severe illness.
J Virol; 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28298600
ResumoThe mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and sub-tropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing 8 of the 13 substrate cleavage sites by dengue NS2B-S3 protease. With no available direct-acting antiviral targeting DENV, NS2-S3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity to DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue protease. The design was based on two aprotinin loops, and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 µM against DENV3 WT protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. Viruses of the flaviviral family, including DENV and Zika viruses transmitted by , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, toward achieving better specificity and lower hydrophilicity compared to current scaffolds in the design of anti-flaviviral inhibitors.
J Virol; 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28298606
ResumoRobust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased ß-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5' adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of mechanistic target of rapamycin complex 1 (mTORC1), decreases in DENV-infected cells and is inversely correlated to lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells, and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling, as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy. Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores resulting in enhanced ß-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.
PLoS One; 12(3): e0173673, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28301523