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.

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.

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.

Ullrich-Turner syndrome: relevance of searching for Y chromosome fragments.

Forty consecutive patients with Ullrich-Turner syndrome (UTS) were followed-up and investigated for the presence of Y chromosome fragments in their genomes. We used the polymerase chain reaction (PCR) to detect SRY (sex-determining region on the Y chromosome) and the sequence-tagged sites (STS) sY57, sY59, sY85, sY94, sY124 and sY157--which correspond to regions 3C (sY57 and 59), 5C, 5G, 5P, and 6F, respectively, of the Y chromosome--searching for Y fragments that could bear the putative locus (loci) for gonadoblastoma (GBY). It has been shown that the presence of GBY greatly increases the risk of dysgenic gonads to undergo malignant transformation. Among our 40 patients, we found Y-derived sequences--including SRY and the region spanning from sY57 to sY94--in two. These two patients had a marker chromosome detected by conventional cytogenetic analysis (45,X/46,X + mar). Their gonads were excised and found to be streaks. In one of the patients, we found foci of primitive sex cords (amidst the gonadal stroma), oviducts and Wolffian remnants. Fluorescence in situ hybridization (FISH) did not show Y chromosome material in her gonad-derived fibroblasts. The other girl had hyperplastic Leydig cells in the gonadal stroma, oviducts and Wolffian remnants, with signs of epididymal differentiation. PCR assays performed on DNA extracted from paraffin-embedded gonadal tissue were negative for SRY sequences in both patients. These findings show that all UTS patients should be examined for Y chromosome material, and that positive cases should have their dysgenic gonads excised due to the high risk of malignancy.