Phylogeography and evolutionary history of dengue virus type 3

In this study, we revisited the phylogeography of the three of major DENV-3 genotypes and estimated its rate of evolution, based on the analysis of the envelope (E) gene of 200 strains isolated from 31 different countries around the world over a time period of 50 years (1956-2006). Our phylogenetic analysis revealed a geographical subdivision of DENV-3 population in several country-specific clades. Migration patterns of the main DENV-3 genotypes showed that genotype I was mainly circumspect to the maritime portion of Southeast-Asia and South Pacific, genotype 11 stayed within continental areas in South-East Asia, while genotype III spread across Asia, East Africa and into the Americas. No evidence for rampant co-circulation of distinct genotypes in a single locality was found, suggesting that some factors, other than geographic proximity, may limit the continual dispersion and reintroduction of new DENV-3 variants. Estimates of the evolutionary rate revealed no significant differences among major DENV-3 genotypes. The mean evolutionary rate of DENV-3 in areas with long-term endemic transmissions (i.e., Indonesia and Thailand) was similar to that observed in the Americas, which have been experiencing a more recent dengue spread. We estimated the origin of DENV-3 virus around 1890, and the emergence of current diversity of main DENV-3 genotypes between the middle 1960s and the middle 1970s, coinciding with human population growth, urbanization, and massive human movement, and with the description of the first cases of DENV-3 hemorrhagic fever in Asia. (C) 2008 Elsevier B.V. All rights reserved.

New Genotype of Dengue Type 3 Virus Circulating in Brazil and Colombia Showed a Close Relationship to Old Asian Viruses

Dengue type 3 genotype V viruses have been recently detected in Brazil and Colombia. In this study, we described another Brazilian isolate belonging to this genotype. Phylogenetic analysis including dengue type 3 viruses isolated worldwide showed that Brazilian and Colombian viruses were closely related to viruses isolated in Asia more than two decades ago. The characteristic evolutionary pattern of dengue type 3 virus cannot explain the close similarity of new circulating viruses with old viruses. Further studies are needed to confirm the origin of the new dengue type III genotype circulating in Brazil and Colombia.

Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the reintroduction of DENV2 in the coast of Sao Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the Sao Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of Sao Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas.

Mosquitoes infected with dengue viruses in Brazil

Dengue epidemics have been reported in Brazil since 1985. The scenery has worsened in the last decade because several serotypes are circulating and producing a hyper-endemic situation, with an increase of DHF/DSS cases as well as the number of fatalities. Herein, we report dengue virus surveillance in mosquitoes using a Flavivirus genus-specific RT-Hemi-Nested-PCR assay. The mosquitoes (Culicidae, n = 1700) collected in the Northeast, Southeast and South of Brazil, between 1999 and 2005, were grouped into 154 pools. Putative genomes of DENV-1, -2 and -3 were detected in 6 mosquito pools (3.8%). One amplicon of putative DENV-1 was detected in a pool of Haemagogus leucocelaenus suggesting that this virus could be involved in a sylvatic cycle. DENV-3 was found infecting 3 pools of larvae of Aedes albopictus and the nucleotide sequence of one of these viruses was identified as DENV-3 of genotype III, phylogenetically related to other DENV-3 isolated in Brazil. This is the first report of a nucleotide sequence of DENV-3 from larvae of Aedes albopictus.

Evolutionary history of Dengue virus type 4: Insights into genotype phylodynamics

Dengue virus type 4 (DENV-4) circulates in tropical and subtropical countries from Asia and the Americas. Despite the importance of dengue virus distribution, little is known about the worldwide viral spread. Following a Bayesian phylogenetic approach we inferred the evolutionary history of 310 isolates sampled from 37 countries during the time period 1956-2008 and the spreading dynamics for genotypes I and II. The region (tropical rainforest biome) comprised by Malaysia-Thailand was the most likely ancestral area from which the serotype has originated and spread. Interestingly, cross-correlation analysis on demographic time series with the Asian sequences showed a statistically significant negative correlation that could be suggestive of competition among genotypes within the same serotype. (C) 2011 Elsevier B.V. All rights reserved.

Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein

The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coil are described The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E coli but not the denatured form or the same protein submitted to a different refolding condition Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods. (C) 2010 Elsevier B V All rights reserved

Low genetic diversities of rabies virus populations within different hosts in Brazil

The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are suggested to have arisen in association with the structural and functional constraints operating on the virus protein and the infection strategies employed by RABV within infected hosts to avoid strong selection by the immune response. In order to investigate the relationship between the genetic characteristics of RABV populations within hosts and the virus evolution, the present study examined the genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of 0-0.95 x 10(-3)). In addition, amino acid mutations were randomly distributed over the entire region of the G protein, and the nonsynonymous/synonymous rate ratios (d(N)/d(S)) for the G protein gene were less than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the evolutionary patterns of the virus. (C) 2009 Elsevier B.V. All rights reserved.

Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.

Dengue and dengue hemorrhagic fever among adults: Clinical outcomes related to viremia, serotypes, and antibody response

Background. Clinical manifestations of dengue vary in different areas of endemicity and between specific age groups, whereas predictors of outcome have remained controversial. In Brazil, the disease burden predominantly affects adults, with an increasing trend toward progression to dengue hemorrhagic fever (DHF) noted. Methods. A cohort of adults with confirmed cases of dengue was recruited in central Brazil in 2005. Patients were classified according to the severity of their disease. Associations of antibody responses, viremia levels (as determined by real-time polymerase chain reaction [PCR]), and serotypes (as determined by multiplex PCR) with disease severity were evaluated. Results. Of the 185 symptomatic patients > 14 years of age who had a confirmed case of dengue, 26.5% and 23.2% were classified as having intermediate dengue fever (DF)/ DHF (defined as internal hemorrhage, plasma leakage, manifested signs of shock, and/ or thrombocytopenia [platelet count, <= 50,000 platelets/mm(3)]) and DHF, respectively. The onset of intermediate DF/ DHF and DHF occurred at a late stage of disease, around the period of defervescence. Patients with DHF had abnormal liver enzyme levels, with a > 3-fold increase in aspartate aminotransferase level, compared with the range of values considered to be normal. Overall, 65% of patients presented with secondary infections with dengue virus, with such infection occurring in similar proportions of patients in each of the 3 disease category groups. Dengue virus serotype 3 (DV3) was the predominant serotype, and viremia was detected during and after defervescence among patients with DHF or intermediate DF/ DHF. Conclusions. Viremia was detected after defervescence in adult patients classified as having DHF or intermediate DF/ DHF. Secondary infection was not a predictor of severe clinical manifestation in adults with infected with the DV3 serotype.

Staphylococcus hominis subsp novobiosepticus strains causing nosocomial bloodstream infection in Brazil

To report the isolation of six Staphylococcus hominis subsp. novobiosepticus (SHN) strains from hospitalized patients with bloodstream infections in two Brazilian hospitals and to characterize their susceptibility profile to several antimicrobials. Species identification was performed by biochemical methods and sodA gene sequencing. The MICs of antimicrobials were determined by broth and agar dilution methods and by Etest. Isolates were typed by PFGE and PCR amplification was used to detect the ccr gene complex and the mec class. Morphometric evaluation of cell wall was performed by transmission electron microscopy (TEM). Susceptibility profiles indicated that the majority of isolates (five) were multidrug-resistant. Overlapping and multiplex PCR showed that five out of the six strains harboured SCCmec type III with class A mec and type 3 ccr. The initial vancomycin MIC value of 4 mg/L for these strains increased to 16-32 mg/L after growth for 10 days in BHI broth supplemented with this antimicrobial. TEM indicated that vancomycin resistance was associated with cell wall thickening and to another mechanism not fully elucidated. Only one SHN strain was oxacillin- and vancomycin-susceptible. The nosocomial infections in at least five of the patients from both hospitals were caused by a single clone of SHN. It is very important to consider SHN strains as the cause of nosocomial infections. The clinical implications resulting from the pattern of multidrug resistance in these strains may be complicated by the emergence of vancomycin resistance.

Impact of pressure profile and duration of recruitment maneuvers on morphofunctional and biochemical variables in experimental lung injury

Objective: To investigate the effects of the rate of airway pressure increase and duration of recruitment maneuvers on lung function and activation of inflammation, fibrogenesis, and apoptosis in experimental acute lung injury. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: Thirty-five Wistar rats submitted to acute lung injury induced by cecal ligation and puncture. Interventions: After 48 hrs, animals were randomly distributed into five groups (seven animals each): 1) nonrecruited (NR); 2) recruitment maneuvers (RMs) with continuous positive airway pressure (CPAP) for 15 secs (CPAP15); 3) RMs with CPAP for 30 secs (CPAP30); 4) RMs with stepwise increase in airway pressure (STEP) to targeted maximum within 15 secs (STEP15); and 5) RMs with STEP within 30 secs (STEP30). To perform STEP RMs, the ventilator was switched to a CPAP mode and positive end-expiratory pressure level was increased stepwise. At each step, airway pressure was held constant. RMs were targeted to 30 cm H(2)O. Animals were then ventilated for 1 hr with tidal volume of 6 mL/kg and positive end-expiratory pressure of 5 cm H(2)O. Measurements and Main Results: Blood gases, lung mechanics, histology (light and electronic microscopy), interleukin-6, caspase 3, and type 3 procollagen mRNA expressions in lung tissue. All RMs improved oxygenation and lung static elastance and reduced alveolar collapse compared to NR. STEP30 resulted in optimal performance, with: 1) improved lung static elastance vs. NR, CPAP15, and STEP15; 2) reduced alveolar-capillary membrane detachment and type 2 epithelial and endothelial cell injury scores vs. CPAP15 (p < .05); and 3) reduced gene expression of interleukin-6, type 3 procollagen, and caspase 3 in lung tissue vs. other RMs. Conclusions: Longer-duration RMs with slower airway pressure increase efficiently improved lung function, while minimizing the biological impact on lungs. (Crit Care Med 2011; 39:1074-1081)

Recruitment maneuver in experimental acute lung injury: The role of alveolar collapse and edema

Objective: In acute lung injury, recruitment maneuvers have been used to open collapsed lungs and set positive end-expiratory pressure, but their effectiveness may depend on the degree of lung injury. This study uses a single experimental model with different degrees of lung injury and tests the hypothesis that recruitment maneuvers may have beneficial or deleterious effects depending on the severity of acute lung injury. We speculated that recruitment maneuvers may worsen lung mechanical stress in the presence of alveolar edema. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: Thirty-six Wistar rats randomly divided into three groups (n = 12 per group). Interventions: In the control group, saline was intraperitoneally injected, whereas moderate and severe acute lung injury animals received paraquat intraperitoneally (20 mg/kg [moderate acute lung injury] and 25 mg/kg [severe acute lung injury]). After 24 hrs, animals were further randomized into subgroups (n = 6/each) to be recruited (recruitment maneuvers: 40 cm H(2)O continuous positive airway pressure for 40 secs) or not, followed by 1 hr of protective mechanical ventilation (tidal volume, 6 mL/kg; positive end-expiratory pressure, 5 cm H(2)O). Measurements and Main Results: Only severe acute lung injury caused alveolar edema. The amounts of alveolar collapse were similar in the acute lung injury groups. Static lung elastance, viscoelastic pressure, hyperinflation, lung, liver, and kidney cell apoptosis, and type 3 procollagen and interleukin-6 mRNA expressions in lung tissue were more elevated in severe acute lung injury than in moderate acute lung injury. After recruitment maneuvers, static lung elastance, viscoelastic pressure, and alveolar collapse were lower in moderate acute lung injury than in severe acute lung injury. Recruitment maneuvers reduced interleukin-6 expression with a minor detachment of the alveolar capillary membrane in moderate acute lung injury. In severe acute lung injury, recruitment maneuvers were associated with hyperinflation, increased apoptosis of lung and kidney, expression of type 3 procollagen, and worsened alveolar capillary injury. Conclusions: In the presence of alveolar edema, regional mechanical heterogeneities, and hyperinflation, recruitment maneuvers promoted a modest but consistent increase in inflammatory and fibrogenic response, which may have worsened lung function and potentiated alveolar and renal epithelial injury. (Crit Care Med 2010; 38: 2207-2214)

Dipeptidyl peptidase IV inhibition downregulates Na(+)-H(+) exchanger NHE3 in rat renal proximal tubule

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO(2))]-pyrrolidide (I40; 60 mg center dot kg(-1)center dot day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[ Z(NO(2))]-OH served as controls. Na(+) -H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output ( 67 +/- 9%; P < 0.01), fractional sodium excretion ( 63 +/- 7%; P < 0.01), as well as lithium clearance ( 81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO(3) reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) -H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Pt(II) and Ag(I) complexes with acesulfame: Crystal structure and a study of their antitumoral, antimicrobial and antiviral activities

Two new complexes of platinum(II) and silver(I) with acesulfame were synthesized. Acesulfame is in the anionic form acesulfamate (ace). The structures of both complexes were determined by X-ray crystallography. For K(2)[PtCl(2)(ace)(2)] the platinum atom is coordinated to two Cl(-) and two N-acesulfamate atoms forming a trans-square planar geometry. Each K(+) ion interacts with two oxygen atoms of the S(=O)(2) group of each acesulfamate. For the polymeric complex [Ag(ace)](n) the water molecule bridges between two crystallographic equivalent Agl atoms which are related each other by a twofold symmetry axis. Two Agl atoms, related to each other by a symmetry centre, make bond contact with two equivalent oxygen atoms. These bonds give rise to infinite chains along the unit cell diagonal in the ac plane. The in vitro cytotoxic analyses for the platinum complex using HeLa (human cervix cancer) cells show its low activity when compared to the vehicle-treated cells. The Ag(I) complex submitted to in vitro antimycobacterial tests, using the Microplate Alamar Blue (MABA) method, showed a good activity against Mycobacterium tuberculosis, responsible for tuberculosis, with a minimal inhibitory concentration (MIC) value of 11.6 mu M. The Ag(I) complex also presented a promising activity against Gram negative (Escherichia colt and Pseudomonas aeruginosa) and Gram positive (Enterococcus faecalis) microorganisms. The complex K(2)[PtCl(2)(ace)(2)] was also evaluated for antiviral properties against dengue virus type 2 (New Guinea C strain) in Vero cells and showed a good inhibition of dengue virus type 2 (New Guinea G strain) replication at 200 mu M, when compared to vehicle-treated cells. (C) 2010 Elsevier Inc. All rights reserved.