Estudo de variáveis virais, clínicas e imunológicas associadas ao Dengue vírus tipo 2 circulante no Brasil

Made available in DSpace on 2017-10-30T15:37:12Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) nieli_faria_ioc_dout_2016.pdf: 14533766 bytes, checksum: 05b1745a4877fc77adb250f69573d76a (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Atualmente a dengue é a arbovirose de maior importância mundial e um dos principais problemas de saúde pública no Brasil desde sua introdução na década de 1980. Dengue é uma doença febril aguda e geralmente de evolução benigna podendo evoluir para forma grave quando na presença de distúrbios hemodinâmicos, caracterizados por manifestações hemorrágicas graves, derrames cavitários e choque. Apesar do conhecimento crescente a cerca da doença e seu vetor, a evolução da dengue para quadros clínicos graves não está totalmente esclarecida. Os quatro sorotipos do vírus dengue (DENV) variam em termos de patogenicidade e virulência, embora essas diferenças ainda não sejam bem compreendidas. O entendimento dos mecanismos envolvidos na patogênese da doença e a identificação precoce de marcadores de gravidade constitui um dos principais desafios das pesquisas envolvendo dengue. Desta forma, o objetivo deste estudo foi de analisar os aspectos virais, epidemiológicos, clínicos e imunológicos de infecções causadas por DENV-2 circulantes no Brasil. Para isto, amostras de DENV-2 isoladas de pacientes com diferentes manifestações clínicas e representativas de seis estados brasileiros, de 1990 a 2010 foram sequenciadas. Avaliamos 260 casos confirmados de dengue atendidos nos centros de saúde dos estados do Rio de Janeiro (RJ) e Mato Grosso do Sul (MS) nos anos de 2010 e 2013. Os pacientes foram classificados de acordo com a nova classificação da OMS, 2009 nos grupos: 163 (62.7%) dengue sem sinais de alarme (DSSA), 75 (28,8%) dengue com sinais de alarme (DCSA) e 16 (6.2%) dengue grave (DG). Os resultados obtidos pelo sequenciamento parcial e / ou total do genoma caracterizaram as amostras de DENV-2 brasileiras como pertencentes ao genótipo do Sudeste Asiático e duas linhagens dentro deste genótipo foram identificadas As cepas circulantes na introdução do DENV-2 1990-2003) pertencem à Linhagem I e cepas isoladas após a reintrodução do DENV-2 em 2007 pertencem à Linhagem II. A análise da carga viral das duas linhagens de DENV-2 demonstrou altos títulos em amostras originárias de pacientes graves e do período 2007-2010 (linhagem II) quando comparados com as amostras do período 1990-2003. Ainda, a análise comparativa de epidemias ocorridas no MS em dois períodos distintos (DENV1/2 em 2010 e DENV-4 em 2013) demonstrou que apesar da circulação dos 4 sorotipos de DENV no estado, a reintrodução do DENV-4 não resultou numa maior incidência de casos graves. Por outro lado, pacientes infectados pelo DENV1/2 (2010) apresentaram menores contagens de plaquetas, maiorfrequência de hospitalização e casos graves quando comparados com os pacientes infectados pelo DENV-4 (2013). A proteína não estrutural NS1 é frequentemente associada com casos graves de dengue e, de fato, altos níveis circulantes de NS1 foram encontrados em pacientes infectados pelos sorotipos DENV1/2 e apresentando formas mais graves da doença. Um caso fatal decorrente de hepatite fulminante, uma rara complicação da infecção pelo DENV, com diagnostico confirmado de infecção pelo DENV-2 (linhagem II) foi reportado em um paciente sem histórico prévio de doença hepática. Por fim, avaliamos a resposta imunológica inata in vitro induzida pelas duas linhagens de DENV-2 determinando as taxas de replicação viral e a indução de um perfil de citocinas após infecção de células dendríticas derivadas de monócitos humanos realizando análises comparativas Os resultados demonstraram que ambas linhagens de DENV-2 foram capazes de infectar células dendriticas humanas e não foram observadas diferenças quanto a taxa de replicação viral ou produção de citocinas quando estas foram comparadas entre si. Interessantemente, sobrenadantes das células dendriticas infectadas com linhagem II apresentaram altos níveis da proteína NS1. Estes resultados em conjunto sugerem que a filogenia e caracterização molecular das cepas de DENV circulantes podem predizer o impacto de cepas virulentas na população. Além disso, a identificação de biomarcadores, relacionados tanto ao hospedeiro quanto ao vírus, poderá contribuir na prevenção das formas graves da doençaCurrently dengue is the most important arbovirus of world and is the main public health problems in Brazil since its introduction in the 1980s. Dengue is an acute febrile illness usually benign and may progress to severe form in the presence of hemodynamic disturbances characterized by severe hemorrhagic manifestations, cavitary stroke and shock. Despite the increasing knowledge about the disease and its vector, the evolution of dengue to severe clinical forms is not fully understood. The four DENV serotypes vary in terms of pathogenicity and virulence, although these differences are poorly understood. Understanding mechanisms involved in the pathogenesis of disease as well as early markers of severity identification is one the main challenges of dengue research. In this context, the aim of this study was to analyze virus, epidemiological, clinical as well as immunological aspects of infections caused by DENV-2 serotypes circulating in Brazil. For this, DENV-2 strains isolated from patients with different clinical manifestations and representative of six Brazilian states, from 1990-2010 were sequenced. We evaluate 260 confimed dengue cases that were treated at health centers in Rio de Janeiro (RJ) and Mato Grosso do Sul (MS) states in 2010 and 2103. Patients were classified according to the new dengue classification WHO 2009 in the following groups: 163 (62.7%) dengue without warning signs, 75 (28,8%) dengue with warning signs, and 16 (6.2%) severe dengue The results obtained by partial sequencing and / or total genome characterized samples of DENV-2 Brazilian as belonging to the genotype of Southeast Asia and two lineages within this genotype were identified. Strains circulating prior DENV-2 emergence (1990-2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Analysis of viral loads of both DENV-2 lineages showed higher titers in samples originated from severe patients 2007-2010 (lineage II) when compared with from 1990-2003. Also, comparative analysis of epidemics occurred in MS during two different periods (DENV1 / 2 in 2010 and DENV-4 in 2013) showed that despite circulation of four serotypes in the state, the reintroduction of DENV-4 did not result in a higher incidence of severe cases. Moreover, DENV1/2 2010 infected patients showed lower platelets counts, higher frequency of hospitalization and severe cases as compared with patients infected with DENV-4 (2013). The nonstructural protein NS1 is often associated with severe cases of dengue and, indeed, high circulating levels of NS1 were found in DENV1/2 infected patients and those presenting more severe forms of disease. A fatal case evolving fulminant hepatitis associated with DENV-2 (lineage II), a rare complication of dengue infection, was reported in patient without a previous history of liver disease Finally, we evaluated the innate immune response induced by both DENV-2 lineages determining viral replication rates and the induction of a cytokine profile after infection of dendritic cells derived from human monocytes in vitro performing comparative analyses. Our results showed that both DENV-2 lineages were able to infect human dendritic cells and no difference was found in the viral replication ratio or in the cytokine production after comparative analysis. Interestingly, infected dendritic cells supernatants from lineage II exhibited high levels of NS1 protein. These results together suggest that the phylogeny and molecular characterization of circulating DENV strains can predict the impact of virulent strains in the population. Furthermore, the identification of biomarkers, related to both host and virus will contribute in the prevention of severe forms of diseas

Histopathological and ultrastructural studies of lung tissue of mice reinfected with dengue virus serotypes 1 or 2

Submitted by Sandra Infurna ([email protected]) on 2018-08-27T14:09:09Z No. of bitstreams: 1 enderson_silva_etal_IOC_2009.pdf: 405933 bytes, checksum: b6dbfa7e1119742c0d02559616ace815 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-08-27T14:17:42Z (GMT) No. of bitstreams: 1 enderson_silva_etal_IOC_2009.pdf: 405933 bytes, checksum: b6dbfa7e1119742c0d02559616ace815 (MD5)Made available in DSpace on 2018-08-27T14:17:42Z (GMT). No. of bitstreams: 1 enderson_silva_etal_IOC_2009.pdf: 405933 bytes, checksum: b6dbfa7e1119742c0d02559616ace815 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Departamento de Histologia e Embriologia,. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Histological and ultrastructural alterations in lung of BALB/c mice reinfected with heterologous DENV by the intravenous route with non-neuroadapted dengue viruses serotypes 1 and 2 were analyzed. The lung samples were processed following the standard techniques of photonic and transmission electron microscopy. Morphological studies showed breakdown of alveolocapilar barrier leading to alveolitis, focal zones of collapse, and intraalveolar hemorrhage. Inside alveolar septa, congested capillaries exhibited neutrophils and platelets. Alveolar capillary endothelial cells exhibited aspects of activation with exuberant phylopodia and intracytoplasmic vesicles and vacuoles. Morphometrical analyses demonstrated an increase of the surface density of interalveolar septa in all reinfected mice while alveolar space density was decreased. All these morphometrical data corroborated the histological features emphasizing the alveolocapilar breakdrown in dengue infected animals. Important is the fact that reinfection leads to intraalveolar fibrogenesis as demonstrated by histomorphometry and ultrastructural studies. DENV particles, antigens and RNA were observed 72 hours post-reinfection in mosquito cells (C6/36) inoculated with sera of the animals. The morphological alterations observed in lungs were similar to the observed in human cases of dengue fever and dengue hemorrhagic fever. The present study demonstrates that the BALB/c mice, during the secondary infection by a heterologous serotype of DENV, develop morphological alterations in lung tissue more severe than those observed in the primary infection

Morphological studies of hepatic tissue of mice reinfected with dengue virus serotypes 1 or 2

Submitted by Sandra Infurna ([email protected]) on 2018-04-26T10:48:54Z No. of bitstreams: 1 debora_vieira_etal_IOC_2011.pdf: 1565061 bytes, checksum: 0712963f06509eec67a0a67bacaa8696 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-04-26T10:59:30Z (GMT) No. of bitstreams: 1 debora_vieira_etal_IOC_2011.pdf: 1565061 bytes, checksum: 0712963f06509eec67a0a67bacaa8696 (MD5)Made available in DSpace on 2018-04-26T10:59:30Z (GMT). No. of bitstreams: 1 debora_vieira_etal_IOC_2011.pdf: 1565061 bytes, checksum: 0712963f06509eec67a0a67bacaa8696 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biológicas. Departamento de Histologia e Embriologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Morfologia e Morfogênese Viral. Rio de Janeiro, RJ. Brasil.Histopathological and ultrastructural aspects of liver of non-neuroadapted BALB/c mice reinfected by the intravenous route with dengue virus serotypes 1 and 2 were presented. The hepatic tissue was processed following the standard techniques for photonic and transmission electron microscopy. Morphological studies showed vacuolization of hepatocytes, inflammatory cells inside sinusoidal capillaries, enlargement of sinusoidal capillaries, foci of hemorrhage inside the interstitium, increase of surface density of reticular fibres, increase of numerical density of sinusoidal cells, decrease of surface density of hepatocytes, edema in the peri centrolobular vein space and presence of phyllopods and pseudopod-like extensions in endothelial cells. DENV particles, virus antigens and DENV RNA were observed in mosquito cells (C6/36) inoculated with sera of the animals 72 hours post-reinfection. The hepatic alterations observed in our experimental model were similar to those observed in human cases of dengue hemorrhagic fever. The present study shows that BALB/c mice reinfected with a heterologous serotype of DENV develop more severe lesions than those observed in mice in primary infection

Subsets of Cytokines and Chemokines from DENV-4-Infected Patients Could Regulate the Endothelial Integrity of Cultured Microvascular Endothelial Cells

Introduction: It is a consensus that inflammatory mediators produced by immune cells contribute to changes in endothelial permeability in dengue. We propose to relate inflammatory mediators seen in dengue patients with the in vitro alteration of endothelial cells (ECs) cultured with serum from these patients. Methods: Patients with mild (DF) to moderate and severe dengue (DFWS/Sev) were selected. ELISA quantified inflammatory mediators. Expression of adhesion molecules and CD147 were evaluated in the ECs cultured with the patient’s serum by flow cytometry. We assessed endothelial permeability by measuring transendothelial electrical resistance in cocultures of ECs with patient serum. Results: Dengue infection led to an increase in inflammatory mediators—the IL-10 distinguished DF from DFWS/Sev. There were no changes in CD31, CD54, and CD106 but decreased CD147 expression in ECs. DFWS/Sev sera induced a greater difference in endothelial permeability than DF sera. Correlation statistical test indicated that low IL-10 and IFN-γ and high CCL5 maintain the integrity of ECs in DF patients. In contrast, increased TNF, IFN-γ, CXCL8, and CCL2 maintain EC integrity in DFWS/Sev patients. Conclusions: Our preliminary data suggest that a subset of inflammatory mediators may be related to the maintenance or loss of endothelial integrity, reflecting the clinical prognosis

Plasma lipidome profiling of newborns with antenatal exposure to Zika virus.

The 2015-2016 Zika virus (ZIKV) outbreak in Brazil was remarkably linked to the incidence of microcephaly and other deleterious clinical manifestations, including eye abnormalities, in newborns. It is known that ZIKV targets the placenta, triggering an inflammatory profile that may cause placental insufficiency. Transplacental lipid transport is delicately regulated during pregnancy and deficiency on the delivery of lipids such as arachidonic and docosahexaenoic acids may lead to deficits in both brain and retina during fetal development. Here, plasma lipidome profiles of ZIKV exposed microcephalic and normocephalic newborns were compared to non-infected controls. Our results reveal major alterations in circulating lipids from both ZIKV exposed newborns with and without microcephaly relative to controls. In newborns with microcephaly, the plasma concentrations of hydroxyoctadecadienoic acid (HODE), primarily as 13-HODE isomer, derived from linoleic acid were higher as compared to normocephalic ZIKV exposed newborns and controls. Total HODE concentrations were also positively associated with levels of other oxidized lipids and several circulating free fatty acids in newborns, indicating a possible plasma lipidome signature of microcephaly. Moreover, higher concentrations of lysophosphatidylcholine in ZIKV exposed normocephalic newborns relative to controls suggest a potential disruption of polyunsaturated fatty acids transport across the blood-brain barrier of fetuses. The latter data is particularly important given the neurocognitive and neurodevelopmental abnormalities observed in follow-up studies involving children with antenatal ZIKV exposure, but normocephalic at birth. Taken together, our data reveal that plasma lipidome alterations associated with antenatal exposure to ZIKV could contribute to identification and monitoring of the wide spectrum of clinical phenotypes at birth and further, during childhood

Twenty Years of DENV-2 Activity in Brazil: Molecular Characterization and Phylogeny of Strains Isolated from 1990 to 2010

<div><p>In Brazil, dengue has been a major public health problem since its introduction in the 1980s. Phylogenetic studies constitute a valuable tool to monitor the introduction and spread of viruses as well as to predict the potential epidemiological consequences of such events. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, viral strains isolated from patients presenting different disease manifestations (<i>n</i> = 34), representing six states of the country, from 1990 to 2010, were sequenced. Partial genome sequencing (genes C/prM/M/E) was performed in 25 DENV-2 strains and full-length genome sequencing (coding region) was performed in 9 strains. The percentage of similarity among the DENV-2 strains in this study and reference strains available in Genbank identified two groups epidemiologically distinct: one represented by strains isolated from 1990 to 2003 and one from strains isolated from 2007 to 2010. No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease severity has a genetic origin, it is not only due to the differences observed on the E gene. The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease either. The analysis based on the partial and/or complete genome sequencing has characterized the Brazilian DENV-2 strains as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was established that strains circulating prior DENV-2 emergence (1990–2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Furthermore, all DENV-2 strains analyzed presented an asparagine (N) in E₃₉₀, previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin. The percentage of identity of the latter with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 1990s suggests that those viruses did not evolve locally but were due to a new viral Lineage introduction in the country from the Caribbean.</p> </div

Maximum likelihood phylogeny based on the C/prM/M/E genes of 25 Brazilian DENV-2, 1991–2010.

<p>Black circles represent DENV-2 sequences generated in this study. Strains representative from the four genotypes available in Genbank (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) were used for the comparison, DENV-1, DENV-3 and DENV-4 strains were used as outgroup to root the trees. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. DENV strains used were named as follows: Country/strain number/state/year. RJ: Rio de Janeiro, ES: Espirito Santo, CE: Ceará, BA: Bahia, RS: Rio Grande do Sul, RN: Rio Grande do Norte.</p

Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.

<p>Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.</p

Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.

<p>Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.</p