The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model.

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.

Vertical transmission of dengue virus in Aedes aegypti and its role in the epidemiological persistence of dengue in Central and Southern Mexico.

BACKGROUND: Aedes aegypti and Aedes albopictus are the main mosquito species responsible for dengue virus (DENV) transmission to humans in the tropical and subtropical regions of the world. The role of vertical transmission in the epidemiology of dengue and the maintenance of this arbovirus in nature during interepidemic periods remain poorly understood, and DENV vertical transmission could sustain the existence of virus reservoirs within Aedes populations. METHODS: Between April 2011 and October 2012, we monitored vertical transmission of DENV in Ae. aegypti and Ae. albopictus in 9 cities of 4 Mexican states. Aedes eggs were collected in ovitraps, then adults were reared under laboratory conditions and their heads were used to infect C6/36 cells. The presence of flavivirus was detected by immunofluorescence assays (IFA), and DENV infection was confirmed by RT-PCR. RESULTS: About 96% of reared adults were Ae. aegypti and 4.0% were Ae. albopictus. No infection was detected in Ae. albopictus, whereas 54 of 713 (7.8%) of Ae. aegypti pools tested positive. A minimum infection rate (MIR) of 2.52 per 1000 mosquitoes was estimated for Ae. aegypti. DENV-1, DENV-2 & DENV-3 serotypes were detected even during interepidemic periods. CONCLUSIONS: This study reports the evidence of vertical transmission of dengue virus with viral isolation and molecular confirmation in Ae. aegypti eggs collected in four endemic regions of Central and Southern Mexico. Vertical transmission may play a role as a reservoir mechanism during mosquito dormancy in interepidemic periods but with minor participation in transmission during epidemic periods.

TRANSMISSION VERTICALE DU VIRUS DE LA DENGUE CHEZ AEDES AEGYPTI ET SON RÔLE DANS LA PERSISTANCE ÉPIDÉMIOLOGIQUE DE LA DENGUE DANS LE CENTRE ET LE SUD DU MEXIQUE: OBJECTIF: Aedes aegypti et Aedes albopictus sont les principales espèces de moustiques responsables de la transmission du virus de la dengue (DENV) à l'homme dans les régions tropicales et subtropicales du monde. Le rôle de la transmission verticale dans l'épidémiologie de la dengue et le maintien de cet arbovirus dans la nature pendant les périodes d'inter-épidémiques restent mal compris, et la transmission verticale du DENV pourrait maintenir l'existence de réservoirs de virus au sein des populations d'Aedes. Notre objectif était d'évaluer la transmission verticale du DENV au Mexique. MÉTHODES: Entre avril 2011 et octobre 2012, nous avons surveillé la transmission verticale du DENV chez Ae. aegypti et Ae. albopictus dans 9 villes de 4 états mexicains. Les œufs d'Aedes ont été collectés dans des ovitraps, puis les adultes ont été élevés dans des conditions de laboratoire et leur tête a été utilisée pour infecter les cellules C6/36. La présence de flavivirus a été détectée par des tests d'immunofluorescence (IFA) et l'infection par DENV a été confirmée par RT-PCR. RÉSULTATS: 96% des adultes élevés étaient Ae. aegypti et 4,0% étaient Ae. albopictus. Aucune infection n'a été détectée chez Ae. albopictus, alors que 54 des 713 (7,8%) des pools d'Ae. aegypti ont été testés positifs. Un taux d'infection minimum (MIR) de 2,52 pour 1000 moustiques a été estimé pour Ae. aegypti. Les sérotypes DENV-1, DENV-2 et DENV-3 ont été détectés même pendant les périodes inter-épidémiques. CONCLUSIONS: Cette étude rapporte les preuves de transmission verticale du virus de la dengue avec isolement viral et confirmation moléculaire dans les œufs d'Ae. Aegypti collectés dans quatre régions d'endémie du centre et du sud du Mexique. La transmission verticale pourrait jouer un rôle de mécanisme réservoir lors de la dormance des moustiques en période inter-épidémique, mais avec une participation mineure à la transmission en période d'épidémie.

RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.

Role of Monocytes in the Pathogenesis of Dengue.

Diseases caused by dengue virus (DENV) are a major public health problem worldwide, considered one of the infections with more prevalence in tropical and subtropical zones of the world. Despite the intense research in the pathogenesis of DENV, this feature is not well understood. One of the main target cells for DENV infection is monocytes; these phagocytes can play a dual role, since they are essential to control viremia, but they also participate in the induction of tissue damage during DENV infection. Monocytes produce different pro-inflammatory cytokines and chemokines in response to infection, and also mediate endothelial damage. In peripheral blood, monocytes can be divided into three different subpopulations, namely classical, intermediate and non-classical, which differ in frequency, cytokine production, among others. Studies in the last years suggest that non-classical monocytes have higher affinity for microvasculature endothelium compared to other type of monocytes, which implies that they could be more involved in the increase of endothelial permeability observed during DENV infection. This review provides a general view of the role of monocytes and their subpopulations in DENV pathogenesis and its effect in viral replication. Finally, the potential contribution of these phagocytes in the alterations of endothelial permeability is discussed.

Sinalização e metabolismo celulares durante a diferenciação de linfócitos B humanos em secretores de anticorpos na presença do vírus da Dengue in vitro e em pacientes com Dengue / B cell signaling and metabolism during antibody-secreting cell differentiation in the presence of Dengue virus in vitro and in Dengue patients

A Dengue é uma doença viral sistêmica, transmitida por mosquitos, que afeta anualmente cerca de 100 milhões de pessoas em todo o mundo. Causada por quatro sorotipos do vírus da Dengue (DENV), suas manifestações clínicas podem variar de assintomáticas à formas que podem levar a óbito. Curiosamente, os pacientes com Dengue apresentam uma resposta exacerbada das células secretoras de anticorpos (ASCs) no sangue cerca de sete dias após o início dos sintomas. A frequência dessas ASCs induzidas pelo DENV representa mais de 50% de todas as células B circulantes no sangue. Essa quantificação é maior que aquelas encontradas em outras infecções virais, contextos de imunização e até mesmo em pacientes com neoplasias de ASCs. Além disso, a magnitude dessa resposta transitória se correlaciona com a gravidade da doença. Então, como a infecção pelo DENV induz essa resposta enorme? Para responder à essa pergunta, combinamos abordagens in vitro e in silico. Células mononucleares do sangue periférico (PBMCs) obtidas de indivíduos saudáveis foram cultivadas in vitro durante sete dias na presença do DENV ou mitógenos. Após a estimulação pelo DENV, as células B presentes nas PBMCs foram capazes de se diferenciarem em ASCs, tanto fenotipicamente quanto funcionalmente, em magnitude similar àquelas estimuladas com mitógenos. Essa diferenciação demonstrou ser dependente da presença de outras células contidas nas PBMCs, assim como do contato célula-célula. Embora ambos os estímulos tenham sido capazes de induzir a diferenciação de ASCs, eles diferiram metabolicamente e transcricionalmente. PBMCs estimuladas pelo DENV apresentaram um maior consumo de triptofano, associado à maior expressão de IDO1 e IDO2 e maior síntese de quinurenina, bem como maiores expressões de IL-10, BAFF e SYK. Ainda, as concentrações de quinurenina foram positivamente correlacionadas com a enumeração de ASCs nessas culturas. Dados de transcriptoma públicos de pacientes com Dengue também suportam esses achados. Outros flavivírus, como o vírus Zika e a cepa vacinal da Febre Amarela não foram capazes de induzir a mesma magnitude de diferenciação das células B em ASCs in vitro. Tão pouco apresentaram correlação entre a enumeração de ASCs e a síntese de quinurenina. Por fim, através da construção de uma hipotética via de diferenciação de células B em ASCs durante infecção pelo DENV, através da combinação de dados da literatura e transcriptomas públicos, demonstramos que moléculas relacionadas à via do STAT3 (IL-10, IL-6, IRF4 e BLIMP1) estão mais expressas nos pacientes infectados e moléculas que respondem aos sinais de cálcio (Calcineurina, NFATC1, DOK3 e GRB2) estão menos expressas nos pacientes infectados. Esses dados proporcionam um melhor entendimento da resposta de células B durante a infeção pelo DENV, particularmente sobre como o metabolismo e a sinalização das células B estão conectados nesse processo

Dengue is a mosquito-borne viral disease that affects annually about 100 million people worldwide. Caused by four Dengue virus (DENV) serotypes, it ranges from asymptomatic to life threatening forms. Curiously, Dengue patients present an exacerbated blood antibody-secreting cell (ASCs) response around seven days after the symptoms onset. The frequency of those DENV-induced ASCs represents more than 50% of all circulating blood B cells. This is greater than found in others viral infections, immunization contexts and even in ASCs related-leukemia patients. Moreover, the magnitude of that transitory response correlates with the disease severity. So, how does the DENV infection induce this enormous response? In order to answer this question we have combined in vitro and in silico approaches. Peripheral blood mononuclear cells (PBMC) obtained from healthy individuals were cultured in vitro during seven days in the presence of DENV or mitogens. Upon the DENV stimulation, PBMC-contained B cells were able to differentiate phenotypically and functionally into ASCs, both phenotypically and functionally, in a similar magnitude than mitogen-stimulated cells. This differentiation was demonstrated to be dependent of the presence of the remaining PBMCs, as well as of the cell-cell contact. Although both stimuli were able to induce the ASCs differentiation, they differed metabolically and transcriptionally. DENV-stimulated PBMCs showed higher tryptophan consumption, associated with higher IDO1 and IDO2 expression and higher kynurenine synthesis, as well as higher IL-10, BAFF and SYK expressions compared to mitogen-exposed counterparts. Additionally, the kynurenine concentrations were positively correlated with the ASCs-enumeration in those cultures. Public transcriptome data supports these findings as well. Other flaviviruses, such as Zika virus and the attenuated vaccine Yellow Fever were not able to induce the same magnitude of ASCs differentiation in vitro. Hence, they did not present a correlation between the number of generated ASCs and the supernatant kynurenine levels. Based on the combination of the literature and public transcriptome data, we have constructed a hypothetical B cell differentiation pathway that might be occurring during DENV infection. It displays that STAT3 pathway-related molecules (IL-10, IL-6, IRF4 and BLIMP1) are more expressed in Dengue patients and molecules that respond to calcium signals (Calcineurin, NFATC1, DOK3 and GRB2) are less expressed in Dengue patients than in control. These data provide a better understanding of the B cell response elicited by DENV infection, particularly about how the B cell metabolism and signaling can be connected into this process

Coinfection with Zika Virus (ZIKV) and Dengue Virus Results in Preferential ZIKV Transmission by Vector Bite to Vertebrate Host.

Background: Several tropical cities are permissive to Aedes aegypti and dengue virus (DENV) endemicity and have allowed for invasion and circulation of Zika virus (ZIKV) in the same areas. People living in arbovirus-endemic regions have been simultaneously infected with ≥2 arboviruses. Methods: A. aegypti mosquitoes from Manaus, the capital city of Amazonas State in Brazil, were coinfected with circulating strains of DENV and ZIKV. The coinfected vectors were allowed to bite BALB/c mice. Results: A. aegypti from Manaus is highly permissive to monoinfection and coinfection with DENV and ZIKV and is capable of cotransmitting both pathogens by bite. Coinfection strongly influences vector competence, favoring transmission of ZIKV to the vertebrate host. Conclusions: This finding suggests that A. aegypti is an efficient vector of ZIKV and that ZIKV would be preferentially transmitted by coinfected A. aegypti. Coinfection in the vector population should be considered a new critical epidemiological factor and may represent a major public health challenge.

Variation in Wolbachia effects on Aedes mosquitoes as a determinant of invasiveness and vectorial capacity.

Wolbachia has been introduced into Aedes aegypti mosquitoes to control the spread of arboviruses, such as dengue, chikungunya and Zika. Studies showed that certain Wolbachia strains (such as wMel) reduce replication of dengue viruses in the laboratory, prompting the release of mosquitoes carrying the bacterium into the field, where vectorial capacity can be realistically assessed in relation to native non-carriers. Here we apply a new analysis to two published datasets, and show that wMel increases the mean and the variance in Ae. aegypti susceptibility to dengue infection when introgressed into Brazil and Vietnam genetic backgrounds. In the absence of other processes, higher mean susceptibility should lead to enhanced viral transmission. The increase in variance, however, widens the basis for selection imposed by unexplored natural forces, retaining the potential for reducing transmission overall.

Pharmacophoric characteristics of dengue virus NS2B/NS3pro inhibitors: a systematic review of the most promising compounds.

Dengue virus (DENV) infection can lead to a wide range of clinical manifestations, including fatal hemorrhagic complications. There is a need to find effective pharmacotherapies to treat this disease due to the lack of specific immunotherapies and antiviral drugs. That said, the DENV NS2B/NS3pro protease complex is essential in both the viral multiplication cycle and in disease pathogenesis, and is considered a promising target for new antiviral therapies. Here, we performed a systematic review to evaluate the pharmacophoric characteristics of promising compounds against NS2B/NS3pro reported in the past 10 years. Online searches in the PUBMED/MEDLINE and SCOPUS databases resulted in 165 articles. Eight studies, which evaluated 3,384,268 molecules exhibiting protease inhibition activity, were included in this review. These studies evaluated anti-dengue activity in vitro and the IC50 and EC50 values were provided. Most compounds exhibited non-competitive inhibition. Cytotoxicity was evaluated in BHK-21, Vero, and LLC-MK2 cells, and the CC50 values obtained ranged from < 1.0 to 780.5 µM. Several groups were associated with biological activity against dengue, including nitro, catechol, halogen and ammonium quaternaries. Thus, these groups seem to be potential pharmacophores that can be further investigated to treat dengue infections.

Estimating the Attack Ratio of Dengue Epidemics under Time-varying Force of Infection using Aggregated Notification Data.

Quantifying the attack ratio of disease is key to epidemiological inference and public health planning. For multi-serotype pathogens, however, different levels of serotype-specific immunity make it difficult to assess the population at risk. In this paper we propose a Bayesian method for estimation of the attack ratio of an epidemic and the initial fraction of susceptibles using aggregated incidence data. We derive the probability distribution of the effective reproductive number, Rt, and use MCMC to obtain posterior distributions of the parameters of a single-strain SIR transmission model with time-varying force of infection. Our method is showcased in a data set consisting of 18 years of dengue incidence in the city of Rio de Janeiro, Brazil. We demonstrate that it is possible to learn about the initial fraction of susceptibles and the attack ratio even in the absence of serotype specific data. On the other hand, the information provided by this approach is limited, stressing the need for detailed serological surveys to characterise the distribution of serotype-specific immunity in the population.

Evidence based community mobilization for dengue prevention in Nicaragua and Mexico (Camino Verde, the Green Way): cluster randomized controlled trial.

OBJECTIVE: To test whether community mobilization adds effectiveness to conventional dengue control. DESIGN: Pragmatic open label parallel group cluster randomized controlled trial. Those assessing the outcomes and analyzing the data were blinded to group assignment. Centralized computerized randomization after the baseline study allocated half the sites to intervention, stratified by country, evidence of recent dengue virus infection in children aged 3-9, and vector indices. SETTING: Random sample of communities in Managua, capital of Nicaragua, and three coastal regions in Guerrero State in the south of Mexico. PARTICIPANTS: Residents in a random sample of census enumeration areas across both countries: 75 intervention and 75 control clusters (about 140 households each) were randomized and analyzed (60 clusters in Nicaragua and 90 in Mexico), including 85,182 residents in 18,838 households. INTERVENTIONS: A community mobilization protocol began with community discussion of baseline results. Each intervention cluster adapted the basic intervention-chemical-free prevention of mosquito reproduction-to its own circumstances. All clusters continued the government run dengue control program. MAIN OUTCOME MEASURES: Primary outcomes per protocol were self reported cases of dengue, serological evidence of recent dengue virus infection, and conventional entomological indices (house index: households with larvae or pupae/households examined; container index: containers with larvae or pupae/containers examined; Breteau index: containers with larvae or pupae/households examined; and pupae per person: pupae found/number of residents). Per protocol secondary analysis examined the effect of Camino Verde in the context of temephos use. RESULTS: With cluster as the unit of analysis, serological evidence from intervention sites showed a lower risk of infection with dengue virus in children (relative risk reduction 29.5%, 95% confidence interval 3.8% to 55.3%), fewer reports of dengue illness (24.7%, 1.8% to 51.2%), fewer houses with larvae or pupae among houses visited (house index) (44.1%, 13.6% to 74.7%), fewer containers with larvae or pupae among containers examined (container index) (36.7%, 24.5% to 44.8%), fewer containers with larvae or pupae among houses visited (Breteau index) (35.1%, 16.7% to 55.5%), and fewer pupae per person (51.7%, 36.2% to 76.1%). The numbers needed to treat were 30 (95% confidence interval 20 to 59) for a lower risk of infection in children, 71 (48 to 143) for fewer reports of dengue illness, 17 (14 to 20) for the house index, 37 (35 to 67) for the container index, 10 (6 to 29) for the Breteau index, and 12 (7 to 31) for fewer pupae per person. Secondary per protocol analysis showed no serological evidence of a protective effect of temephos. CONCLUSIONS: Evidence based community mobilization can add effectiveness to dengue vector control. Each site implementing the intervention in its own way has the advantage of local customization and strong community engagement. TRIAL REGISTRATION: ISRCTN27581154.

An embryonic heart cell line is susceptible to dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

Avaliação do perfil de mediadores séricos e proteínas intraplaquetárias em relação à plaquetopenia em pacientes infectados pelo vírus dengue / Evaluation of serum mediators profile and intraplatelet proteins in respect to thrombocytopenia in patients infected with dengue virus

Plaquetas são fragmentos celulares derivados dos megacariócitos, que desempenham papel na hemostasia, coagulação, angiogênese, inflamação e resposta imune. Na infecção humana pelo Vírus Dengue (DENV), plaquetas constituem uma das populações celulares mais afetadas devido à plaquetopenia e disfunção plaquetária. O objetivo deste trabalho foi investigar a influência de citocinas, quimiocina e fatores de crescimento séricos e de proteínas intraplaquetárias relacionadas à angiogênese, coagulação, regulação da matriz extracelular e inflamação na plaquetopenia de pacientes infectados pelo DENV. Para tal, realizamos: (i) estudo populacional de pacientes e obtenção de soro e plaquetas em 2013, (ii) ensaios multiplex de micrarranjo líquido para quantificação dos níveis séricos de citocinas, quimiocina e fatores de crescimento e (iii) ensaio de determinação do perfil de expressão 55 proteínas intraplaquetárias. Quarenta e três pacientes DENV foram confirmados, com predominância do DENV-4. Independente da forma clínica, pacientes DENV apresentaram níveis séricos elevados de IL-10, TNF-alfa, CXCL8/IL-8, mas não de IL-1beta e IFN-gama quando comparados aos controles sadios. Análises estatísticas demonstraram que níveis de IL-10 e IFN-gama apresentaram correlação, respectivamente inversa e direta com a contagem de plaquetas. Ainda, IL-10 diretamente com leucócitos e linfócitos e TNF-alfa com linfócitos. Vinte e cinco proteínas intraplaquetárias foram quantificadas, mas apenas cinco delas, PDGF-AA, TGF-beta1, HGF, IGFBP-1 e Angiopoetina-1, apresentaram correlação direta com a contagem de plaquetas nos pacientes DENV. A quantificação sérica de PDGF e VEGF demostrou que ambos estavam diminuídos no grupo DENV mais trombocitopênico...

Platelets are cell fragments derived from megakaryocytes, which play a role in hemostasis, coagulation, angiogenesis, inflammation and immune response. In human infection with dengue virus (DENV), platelets are one of the most affected cell populations due to thrombocytopeniaand platelet dysfunction. The objective of this study was to investigate the influence of serum cytokines, chemokines, intraplatelet growth factors and proteins related to angiogenesis, coagulation, regulation of extracellular matrix and inflammation in thrombocytopenia of patientsinfected with DENV. For this purpose, we conducted: (i) population study of patients and obtaining their serum and platelets in 2013, (ii) liquid microarray multiplex assays for quantitationof serum levels of cytokines, chemokine, and growth factors, and (iii) assay for determiningexpression profile of 55 intraplapletelet proteins. Forty-three DENV patients were confirmed, with a predominance of DENV-4. Regardless of type of DENV, levels of IL-10, TNF-alfa, CXCL8 /IL-8, but not IL-1beta and IFN-gama were higher on serum of patients compared to healthy individuals. Statistical analyses showed that levels of IL-10 and IFN-gama presented correlation, respectively, inverse and direct with platelet count. Furthermore, IL-10 was directly correlated with leukocytes, lymphocytes, TNF-alfa and with lymphocytes. Twenty-five intraplatelet proteins were quantified, but only five of them, PDGF-AA, TGF-beta1, HGF, angiopoietin-1 and IGFBP-1 weredirectly correlated with platelet count in DENV patients. Both levels of PDGF and VEGF were decreased in group of DENV thrombocytopenic...

Increased replicative fitness of a dengue virus 2 clade in native mosquitoes: potential contribution to a clade replacement event in Nicaragua.

UNLABELLED: The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua. IMPORTANCE: Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vector-driven evolution of DENV epidemics.

[Selection of heparin-sensitive dengue virus variants in BHK-21 cells]. / Selección de variantes de virus dengue sensibles a la heparina en células BHK-21.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

Selección de variantes de virus dengue sensibles a la heparina en células BHK-21 / Selection of heparin-sensitive dengue virus variants in BHK-21 cells

Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.

Dengue virus: isolation, propagation, quantification, and storage.

Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV-1, -2, -3, and -4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single-stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life-threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV.

Towards uncertainty quantification and inference in the stochastic SIR epidemic model.

In this paper we address the problem of estimating the parameters of Markov jump processes modeling epidemics and introduce a novel method to conduct inference when data consists on partial observations in one of the state variables. We take the classical stochastic SIR model as a case study. Using the inverse-size expansion of van Kampen we obtain approximations for the first and second moments of the state variables. These approximate moments are in turn matched to the moments of an inputed Generic Discrete distribution aimed at generating an approximate likelihood that is valid both for low count or high count data. We conduct a full Bayesian inference using informative priors. Estimations and predictions are obtained both in a synthetic data scenario and in two Dengue fever case studies.

Modelos jerárquicos espacio temporales para mapear riesgos relativos de dengue, en el Municipio Girardot, Estado Aragua, Venezuela / Spatio-temporal hierarchical models for mapping relative risks of dengue in the Municipality of Girardot, Aragua State, Venezuela

Los modelos Bayesianos jerárquicos espaciotemporales han sido usados en el mapeo de enfermedades, estudios de contaminación ambiental, contaminación industrial, entre muchos otros. Bajo esta metodología, los datos están asociados con un punto en una localidad E y con un instante de tiempo t. El objetivo de este trabajo es modelar el riesgo relativo de contraer dengue en el municipio Girardot del estado Aragua, Venezuela, durante el periodo epidemiológico del año 2009. Se proponen tres estructuras de modelos, un Binomial que toma en cuenta la variabilidad en el conteo de la ocurrencia de la enfermedad en las parroquias del municipio. Una segunda propuesta incluye un modelo Binomial como primer nivel de jerarquía, más un segundo nivel que introduce el efecto espacial, el efecto temporal y la interacción espacio-tiempo. Finalmente, un tercer modelo espacial que combina el modelo Poisson en el primer nivel de jerarquía para el número de casos, y en el segundo nivel de jerarquía se relaciona el riesgo relativo con las covariables a través de la función logaritmo más un efecto aleatorio. Los datos fueron recopilados por semanas y clasificados de acuerdo a las parroquias del municipio. Se utilizó el criterio de información de deviancia (DIC) para seleccionar el mejor modelo, resultando el modelo Poisson el más adecuado para representar el riesgo relativo de contraer dengue en la zona bajo estudio, confirmando que los patrones de alto riesgo se encuentran en las parroquias ubicadas al sur y suroeste del municipio Girardot, colindando algunas de ellas con el lago de Valencia.

Hierarchical Bayesian space-time models have been used in the mapping of disease, studies of environmental pollution and industrial pollution, among many others. Under this methodology, the data is associated with point in a locality E and an instant in time t. The aim of this work is to model the relative risk of dengue in Girardot Municipality, Aragua State, Venezuela, during the epidemic period 2009. In that sense, we propose three models. First, a binomial model that measures the variability in the count of occurrence of the disease in the parishes of the municipality. A second model includes the binomial model as a first hierarchical level, plus a second level which introduces the spatial effect, the temporal effect and spacetime interaction. Finally, a third spatial model that follows a Poisson model at the first level of hierarchy for the number of cases, and in the second level of hierarchy relates the relative risk associated with covariates through the logarithm function over a random effect. Data were collected for weeks and classified according to the parishes of the municipality. The Deviance Information Criterion (DIC) was used to select the best model. The Poisson model was best suited to represent the relative risk of contracting dengue in the area under study, showing that high-risk patterns were found in the parishes located in the south and southwest of the Girardot municipality, some of them bordering the lake of Valencia.