Vector Surveillance for Dengue Virus Detection in the Archipelago of Fernando de Noronha, Brazil.

Aedes aegypti (L.) has become an efficient vector of important arboviruses due to its anthropophilic and domiciliary behaviors. Since the 1980s, dengue affects thousands of people every year in Brazil; in Fernando de Noronha (FN), a touristic archipelago, dengue cases have occurred since 2001. Once Ae. aegypti populations are well established in the inhabited areas of FN, the threat of dengue or another arbovirus epidemic is continuously imminent. This study aimed to monitor the DENV serotypes in mosquito samples collected in FN, where at least one resident was clinically diagnosed as dengue patient. Entomological surveillance was conducted in 2011 and 2012. Mosquitoes were sorted by sex and location and were stored in pools. DENV detection was performed using polymerase chain reaction with reverse transcription (RT-PCR) and the Platelia Dengue NS1 Ag. RNA integrity was checked by RT-PCR using rpL8 primers, and the minimum infection rate (MIR) was calculated. In total, 339 pools were analyzed, and only one was positive (DENV-1) by Multiplex RT-PCR (MIR = 1.53). When considering only pools with RNA integrity, the MIR was 2.92. Using the Platelia kit, the MIR was 9.18 (considering all the pools) and 17.54 (only 140 pools with RNA integrity). Our results showed the importance of a constant entomological surveillance in that area, the need to improve storage and transportation protocols, and an endogenous control in the RT-PCR to avoid false-negative results. Finally, our study indicated that the NS1-Ag detection was the most sensitive method and should be used routinely for DENV surveillance in mosquitoes if the serotype identification is not required.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension.

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Immune transcript variations among Aedes aegypti populations with distinct susceptibility to dengue virus serotype 2.

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.

Revising the M235T polymorphism position for the AGT gene and reporting a modifying variant in the Brazilian population with potential cardiac and neural impact.

There is a growing need to curate the overwhelming amount of sequencing data which is available in many public databases. For instance, new information shows that the M235T polymorphism at the angiotensinogen gene (AGT) is actually positioned at the position corresponding to the amino acid 268 and not 235. This polymorphism is filled as rs699 in the NCBI SNP database and results in the synthesis of a threonine (T) instead of a methionine (M). It has been widely studied and associated as an important risk factor for several vascular and neuropsychiatric conditions. We faced this new situation during the targeted sequencing of 360 chromosomes from Brazilian subjects studied for the M235T polymorphism, leading to the identification of a novel variation (rs141900991). This report explores the potential impact of such a dinucleotide variation, which promotes the change of alanine (A) to serine (S) at the AGT protein structure (A237S). Considering the previous M268T variation at the four possible haplotypes combined (MA, MS, TA and TS), we performed a comparative hydrophobicity simulation, using the Kyte-Doolittle algorithm, available at the CLB Bio workbench, in the four possible haplotypes. Additional simulations were performed using the programs PolyPhen, I-Mutant and SIFT, in order to evaluate the pathogenicity of both mutations. The predicted hydrophobicity decreases of a similar magnitude, with both MS and TA haplotypes, but the presence of both variations induces a major decrease in hydrophobicity, suggesting a cumulative effect, with possible modifying effect since that this variation per se would limit the hydrophobicity range and the latter chances in finding significant phenotype differences. A better characterization of this kind of variant is particularly important because the current genome wide scan analyses in complex disorders with cardiac or neural etiology are not generating reliable findings, especially if we consider the huge investment with such approach. Additional and unknown variations like this one, with potential modifying effect, might be more common than previously expected.

Commercial coconut palm as an ecotope of Chagas disease vectors in north-eastern Venezuela.

BACKGROUND & OBJECTIVE: There are few reports of Coccus nucifera (Palmae) infestation by triatomines (Hemiptera, Reduviidae, Triatominae), vectors of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), the causal agent of American Trypanosomiasis. The aim of this study was to determine if this palm is an appropriate ecotope for Rhodnius prolixus and Triatoma maculata, the main vectors in Venezuela. METHODS: Dry and green leaves, humid debris, interfoliaceus meshes and bracts from C. nucifera from north-eastern Venezuela were examined for the presence of triatomines. Samples of the intestinal content of vectors, macerated in isotonic saline solution and haemolymph were examined microscopically for the presence of Trypanosoma spp. The parasites were isolated and characterized using biological parameters and PCR. Triatomine blood meal sources were determined using ELISA. RESULTS: A total of 14 palms were examined in which viable eggs of both species of vectors were found in 13 palms (92.85%). A total of 242 R. prolixus and 144 T. maculata adults were collected, of which 98% of R. prolixus and 70% of T. maculata individuals were infected by T. cruzi (TcI genotype) and 13% of R. prolixus individuals showed a mixed infection with T. rangeli, the other American trypanosome. ELISA testing for possible triatomine blood-meal sources revealed that these vectors are essentially eurytrophic and zoophilic, although R. prolixus also eventually used human blood as a nourishment source. INTERPRETATION & CONCLUSION: The results obtained suggest that C. nucifera is an appropriate vegetal niche for these triatomine species in Venezuela. The presence of this commercial palm may represent a useful environmental bioindicator of risk for Chagas disease.

Patient-based dengue virus surveillance in Aedes aegypti from Recife, Brazil.

BACKGROUND & OBJECTIVES: Dengue is currently one of the most important arthropod-borne diseases and may be caused by four different dengue virus serotypes (DENV-1 to DENV-4), transmitted mainly by Aedes aegypti (Diptera: Culicidae) mosquitoes. With the lack of a dengue vaccine, vector control strategies constitute a crucial mode to prevent or reduce disease transmission. In this context, DENV detection in natural Ae. aegypti populations may serve as a potential additional tool for early prediction systems of dengue outbreaks, leading to an intensification of vector control measures, aimed at reducing disease transmission. In Brazil, this type of surveillance has been performed sporadically by a few groups and has not been incorporated as a routine activity in control programs. This study aimed at detecting DENV in natural Ae. aegypti from Recife, Pernambuco, to check the circulating serotypes and the occurrence of transovarial transmission in local mosquito populations. METHODS: From January 2005 to June 2006, mosquitoes (adults and eggs) were collected in houses where people with clinical suspicion of dengue infection lived at. RNA was extracted from pooled mosquitoes and RT-PCR was performed in these samples for detection of the four DENV serotypes. RESULTS & CONCLUSION: Out of 83 pools of adult mosquitoes collected in the field, nine were positive for DENV: five for DENV-1, two for DENV-2 and two for DENV-3. From 139 pools of adult mosquitoes reared from collected eggs, there were 17 positive pools: three for DENV-1, 10 for DENV-2, and four for DENV-3. These results are discussed in the paper in regard to the local dengue epidemiological data. The conclusions clearly point to the informative power and sensitivity of DENV entomological surveillance and to the importance of including mosquito immature forms in this strategy.

Resistance to the organophosphate temephos: mechanisms, evolution and reversion in an Aedes aegypti laboratory strain from Brazil.

Insecticide resistance is one of the main problems in vector control programs. Because insects have developed resistance to all classes of available chemical insecticides, a proper surveillance and management of resistance in areas where these compounds are being utilized is crucial for the success of control programs. Since the mechanisms and molecular bases of resistance are various, they must be characterized to allow efficient monitoring strategies. Here we report the establishment of an Aedes aegypti strain resistant to temephos, named RecR, selected under laboratory conditions. The parental A. aegypti population was obtained from eggs collected in an area where temephos had been used for 8 years, and presented a baseline resistance ratio (RR) of 7. After 17 generations under selective pressure, the RR has increased to 180. Biochemical assays indicate that metabolic mechanisms are involved on temephos resistance in the selected strain. These experiments showed that, compared to the susceptible colony Rockefeller, RecR present higher activity of glutathione S-transferases (GSTs), alpha- and beta-esterases, and, to a lesser degree, mixed function oxidases (MFO). At the 14th or 17th generations, there was no cross resistance of these insects to deltamethrin, cypermethrin and malathion, while a low resistance level (RR=3) was observed for pyriproxyfen, a juvenile hormone analogue. Experiments on resistance reversal, performed through three different field simulated schemes using the resistant strain, showed that temephos susceptibility can be recovered. The establishment of an A. aegypti colony resistant to temephos is extremely valuable for a deeper understanding of resistance mechanisms and thus for further improvements in control strategies against this vector. With the urgent need on improving methodologies to monitor resistance, molecular studies such as microarrays, and resistant colonies such as RecR will certainly hasten such studies.

Knock-down of REL2, but not defensin A, augments Aedes aegypti susceptibility to Bacillus subtilis and Escherichia coli.

Some components of the Toll and Imd immune signaling pathways have remained conserved between Drosophila and mosquitoes, however, important differences in the way invading microorganisms activate these pathways in these organisms have started to be revealed. In the present study, we have attempted to silence the Aedes aegypti NF-kappaB-like factor REL2, which is analogous to Drosophila Relish, and analyze the effects on mosquito mortality upon infection with a Gram-negative and a Gram-positive bacterium, both containing a DAP-type peptidoglycan, and effects on embryo development. Moreover, we have silenced one of the antimicrobial peptides (AMPs) controlled by REL2, defensin A, a major AMP in A. aegypti, and compared the results on mosquito mortality upon bacterial infection to those obtained with REL2 silencing. Results show that REL2 is crucial for A. aegypti immunity upon infection with Escherichia coli and Bacillus subtilis, corroborating with previous studies on that REL2/Imd in mosquitoes is involved in a generalized antibacterial defense, differently than its analogous in Drosophila. However, defensin A silencing did not cause a significant increase in mortality in infected mosquitoes, indicating that this peptide is not essential in mosquito protection against the two bacteria and that other immune factors controlled by REL2 are playing this role. In regard to embryo development, REL2 knock-down did not cause any significant effect on: the number of laid eggs, number of developed pupae, percent of emerged adults, and ratio between emerged females versus males. A slight decrease in the number of hatched eggs (percent eclosion) was observed in REL2 knock-down mosquitoes, but these observations were inconclusive.

Molecular evidence for apoptosis in microfilariae of Wuchereria bancrofti induced by diethylcarbamazine.

Apoptosis, a programmed cell death, is characterized by chromatin condensation, numerous vacuoles, reduction in cell volume, and endonuclease cleavage DNA degradation detected in gel electrophoresis as nucleosomal ladder. Here we report that diethylcarbamazine induces DNA fragmentation in microfilariae of Wuchereria bancrofti revealed by ligation-mediated polymerase chain reaction and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling at the light and electron transmission level.

Genetic diversity of Brazilian natural populations of Anthonomus grandis Boheman (Coleoptera: Curculionidae), the major cotton pest in the New World.

Twenty-five RAPD loci and 6 isozyme loci were studied to characterize the genetic variability of natural populations of Anthonomus grandis from two agroecosystems of Brazil. The random-amplified polymorphic DNA data disclosed a polymorphism that varied from 52 to 84% and a heterozygosity of 0.189 to 0.347. The index of genetic differentiation (GST) among the six populations was 0.258. The analysis of isozymes showed a polymorphism and a heterozygosity ranging from 25 to 100% and 0.174 to 0.277, respectively. The genetic differentiation (FST) among the populations obtained by isozyme data was 0.544. It was possible to observe rare alleles in the populations from the Northeast region. The markers examined allowed us to distinguish populations from large-scale, intensive farming region (cotton belts) versus populations from areas of small-scale farming

Genetic differentiation of Aedes aegypti (Diptera: Culicidae), the major dengue vector in Brazil.

In 2000, Brazil reported 180,137 cases of dengue, approximately 80% of the total in the Americas. However, little is known about gene flow among the vector populations in Brazil. Random amplified polymorphic DNA (RAPD) was used to study the genetic structure of Aedes aegypti in 15 populations from five states, with a range extending 2,800 km. An analysis of 47 polymorphic RAPD loci estimated gene flow at the macro- (different states) and micro- (different cities) geographical levels. Genetic polymorphism was high (H(S) = 0.274), and high levels of genetic differentiation existed both between different states (G(ST) = 0.317) and between cities or neighborhoods in each state (G(ST) = 0.085-0.265). These values are higher than those described for any other populations of A. aegypti.

Genetic diversity in Brazilian populations of Aedes albopictus.

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations.

Genetic diversity in Brazilian populations of Aedes albopictus

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations