Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Genetic diversity of the E Protein of Dengue Type 3 Virus

Abstract Background Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis. Results Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein. Conclusion Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.

Designing a vaccination strategy against dengue

In this work we propose a mathematical approach to estimate the dengue force of infection, the average age of dengue first infection, the optimum age to vaccinate children against dengue in a routine fashion and the optimum age interval to introduce the dengue vaccine in a mass vaccination campaign. The model is based on previously published models for vaccination against other childhood infections, which resulted in actual vaccination programmes in Brazil. The model was applied for three areas of distinct levels of endemicity of the city of Recife in Northeastern State of Pernambuco, Brazil. Our results point to an optimal age to introduce the dengue vaccine in the routine immunization programme at two years of age and an age interval to introduce a mass vaccination between three and 14 years of age.

Pr��ticas baseadas em evid��ncias publicadas no Brasil: identifica����o e reflex��o na ��rea da prevenção em sa��de humana

Revis��o integrativa de estudos brasileiros sobre pr��ticas baseadas em evid��ncias (PBE) acerca da prevenção em sa��de humana, publicados em peri��dicos Web of Science/JCR, de outubro de 2010 a abril de 2011. O objetivo foi identificar as especialidades que mais realizaram estes estudos, seus enfoques e abordagens metodol��gicas. A partir de crit��rios de inclus��o, foram selecionados 84 trabalhos publicados majoritariamente em peri��dicos de sa��de p��blica, focalizando a aten����o prim��ria e abrangendo tamb��m quest��es cl��nicas e diversas especialidades. Variaram tamb��m os enfoques de prevenção e as abordagens metodol��gicas, predominando a revis��o sistem��tica sem metan��lise. Os resultados indicam que n��o h�� uma ��nica maneira de conceituar e praticar a PBE na prevenção e sua aplica����o pode n��o ser apenas para obten����o de prova irrefut��vel para instrumentalizar a����es de interven����o. Constitui um campo infind��vel de conhecimentos, em constru����o, para an��lise e maior compreens��o de fen��menos em sa��de.

The burden of dengue: Jundia��, Brazil - January 2010

OBJECTIVE: To study the antibody prevalence against dengue in the municipality of Jundia��, S��o Paulo, Brazil, due to the low number of official confirmed autochthonous cases. METHODS: A serological study on dengue infection was conducted during January 2010 and previous reports on dengue and entomological surveillance during that period were reviewed. RESULTS: A prevalence of 7.8% IgG positive (68:876) was found. Furthermore, based on the detection of IgM antibodies in five samples, it was observed that the incidence of dengue in the city at the time of the survey contrasts with the absence of notifications by local health authorities over the same period of time. CONCLUSION: These results highlight the discrepancies between the actual and the detected number of dengue infections, possibly due to significant numbers of asymptomatic infections aggravated by difficulties with dengue clinical diagnosis.

Diretrizes para prevenção e tratamento da osteoporose induzida por glicocorticoide

Os glicocorticoides (GC) s��o prescritos por praticamente todas as especialidades m��dicas, e cerca de 0,5% da popula����o geral do Reino Unido utiliza esses medicamentos. Com o aumento da sobrevida dos pacientes com doen��as reumatol��gicas, a morbidade secund��ria ao uso dessa medica����o representa um aspecto importante que deve ser considerado no manejo de nossos pacientes. As incid��ncias de fraturas vertebrais e n��o vertebrais s��o elevadas, variando de 30%-50% em pessoas que usam GC por mais de tr��s meses. Assim, a osteoporose e as fraturas por fragilidade devem ser prevenidas e tratadas em todos os pacientes que iniciar��o ou que j�� estejam em uso desses esteroides. Diversas recomenda����es elaboradas por v��rias sociedades internacionais t��m sido descritas na literatura, por��m n��o h�� consenso entre elas. Recentemente, o Americam College of Rheumatology publicou novas recomenda����es, por��m elas s��o fundamentadas na FRAX (WHO Fracture Risk Assessment Tool) para analisar o risco de cada indiv��duo e, dessa maneira, n��o podem ser completamente utilizadas pela popula����o brasileira. Dessa forma, a Comiss��o de Osteoporose e Doen��as Osteometab��licas da Sociedade Brasileira de Reumatologia, em conjunto com a Associa����o M��dica Brasileira e a Associa����o Brasileira de Medicina F��sica e Reabilita����o, implementou as diretrizes brasileiras de osteoporose induzida por glicocorticoide (OPIG), baseando-se na melhor evid��ncia cient��fica dispon��vel e/ou experi��ncia de experts. DESCRI����O DO M��TODO DE COLETA DE EVID��NCIA: A revis��o bibliogr��fica de artigos cient��ficos desta diretriz foi realizada na base de dados MEDLINE. A busca de evid��ncia partiu de cen��rios cl��nicos reais, e utilizou as seguintes palavras-chave (MeSH terms): Osteoporosis, Osteoporosis/chemically induced*= (Glucocorticoids= Adrenal Cortex Hormones, Steroids), Glucocorticoids, Glucocorticoids/administration and dosage, Glucocorticoids/therapeutic use, Glucocorticoids/adverse effects, Prednisone/adverse effects, Dose-Response Relationship, Drug, Bone Density/drug effects, Bone Density Conservation Agents/pharmacological action, Osteoporosis/ prevention&control, Calcium, Vitamin D, Vitamin D deficiency, Calcitriol, Receptors, Calcitriol; 1-hydroxycholecalciferol, Hydroxycholecalciferols, 25-Hydroxyvitamin D3 1-alpha-hydroxylase OR Steroid Hydroxylases, Prevention and Control, Spinal fractures/prevention & control, Fractures, Spontaneous, Lumbar Vertebrae/injuries, Lifestyle, Alcohol Drinking, Smoking OR tobacco use disorder, Movement, Resistance Training, Exercise Therapy, Bone density OR Bone and Bones, Dual-Energy X-Ray Absorptiometry OR Absorptiometry Photon OR DXA, Densitometry, Radiography, (Diphosphonates Alendronate OR Risedronate Pamidronate OR propanolamines OR Ibandronate OR Zoledronic acid, Teriparatide OR PTH 1-34, Men AND premenopause, pregnancy, pregnancy outcome maternal, fetus, lactation, breast-feeding, teratogens, Children (6-12 anos), adolescence (13-18 anos). GRAU DE RECOMENDA����O E FOR��A DE EVID��NCIA: A) Estudos experimentais e observacionais de melhor consist��ncia; B) Estudos experimentais e observacionais de menor consist��ncia; C) Relatos de casos (estudos n��o controlados); D) Opini��o desprovida de avalia����o cr��tica, com base em consensos, estudos fisiol��gicos ou modelos animais. OBJETIVO: Estabelecer as diretrizes para a prevenção e o tratamento da OPIG.

Viol��ncia sexual intrafamiliar contra crian��as e adolescentes: entre a prevenção do crime e do dano

OBJETIVOS: compreender as representa����es sociais de membros do Poder Judici��rio acerca da prevenção da viol��ncia sexual intrafamiliar contra crian��as e adolescentes. M��TODOS: pesquisa de abordagem qualitativa que teve como campo de estudo as 1�� e 2�� Varas dos Crimes contra a Crian��a e o Adolescente, no Tribunal de Justi��a de Pernambuco, com participa����o de 17 sujeitos (juiz, assessor, t��cnicos e analistas judici��rios). Observa����o participante, entrevistas semiestruturadas e grupo focal compreenderam as t��cnicas para coleta de dados, que foram analisados por meio da interpreta����o dos sentidos. RESULTADOS: a categoria "cultura penal" emergiu dos discursos, bem como suas subcategorias: "prevenção do crime" e "prevenção do dano". CONCLUS��ES: as representa����es dos sujeitos revelam uma dicotomia, caracterizando o conflito entre a tradi����o do Poder Judici��rio e o direito novo, que abrange os princ��pios da prote����o integral e da prioridade absoluta ��s crian��as e dos adolescentes. O conceito de prevenção da viol��ncia sexual intrafamiliar contra crian��as e adolescentes deve ser ampliado para al��m da mera prevenção do crime. A abordagem do problema por meio da prevenção requer que se incorpore ao Poder Judici��rio uma cultura penal que abarque os princ��pios da prote����o integral e da prioridade absoluta.

Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of Sa��o Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (��DEC205 and ��DCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with ��DEC-NS1 and ��DCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-�� producing cells was higher in ��DEC-NS1 immunized animals. In addition, mice immunized with the ��DEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with ��DCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Dengue virus type 3 adaptive changes during epidemics in S��o Jose de Rio Preto, Brazil, 2006���2007

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of S��o Jos�� de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

Worldwide spread of Dengue virus type 1

BACKGROUND: DENV-1 is one of the four viral serotypes that causes Dengue, the most common mosquito-borne viral disease of humans. The prevalence of these viruses has grown in recent decades and is now present in more than 100 countries. Limited studies document the spread of DENV-1 over the world despite its importance for human health. METHODOLOGY/PRINCIPAL FINDINGS: We used representative DENV-1 envelope gene sequences to unravel the dynamics of viral diffusion under a Bayesian phylogeographic approach. Data included strains from 45 distinct geographic locations isolated from 1944 to 2009. The estimated mean rate of nucleotide substitution was 6.56 �� 10������ substitutions/site/year. The larger genotypes (I, IV and V) had a distinctive phylogenetic structure and since 1990 they experienced effective population size oscillations. Thailand and Indonesia represented the main sources of strains for neighboring countries. Besides, Asia broadcast lineages into the Americas and the Pacific region that diverged in isolation. Also, a transmission network analysis revealed the pivotal role of Indochina in the global diffusion of DENV-1 and of the Caribbean in the diffusion over the Americas. CONCLUSIONS/SIGNIFICANCE: The study summarizes the spatiotemporal DENV-1 worldwide spread that may help disease control.

Risk factors associated with death in Brazilian children with severe dengue: a case-control study

Objective: The purpose of this case-control study was to evaluate risk factors associated with death in children with severe dengue. Methods: The clinical condition of hospitalized patients with severe dengue who died (cases, n���=���18) was compared with that of hospitalized patients with severe dengue who survived (controls, n���=���77). The inclusion criteria for this study were age under 13 years; hospital admission in S��o Luis, northeastern Brazil; and laboratory-confirmed diagnosis of dengue. Results: Severe bleeding (hemoptysis), a defining criterion for dengue severity, was the factor most strongly associated with death in our study. We also found that epistaxis and persistent vomiting, both included as warning signs in the World Health Organization (WHO) classification of dengue, were strongly associated with death. No significant association was observed between any of the laboratory findings and death. Conclusions: The finding that epistaxis and persistent vomiting were also associated with death in children with severe dengue was unexpected and deserves to be explored in future studies. Because intensive care units are often limited in resource-poor settings, any information that can help to distinguish patients with severe dengue with a higher risk to progress to death may be crucial.