The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.

Density and survival rate of Culex quinquefasciatus at Parque Ecologico do Tiete, Sao Paulo, Brazil

Parity rate, gonotrophic cycle length, and density of a Culex quinquefasciatus female population was estimated at the Parque Ecologico do Tiete (PET), Sao Paulo, Brazil. Adult Cx. quinquefasciatus females were collected from vegetation along the edges of a polluted drainage canal with the use of a battery-powered backpack aspirator from September to November 2005 and from February to April 2006. We examined 255 Cx. quinquefasciatus ovaries to establish the parity rate of 0.22 and determined the gonotrophic cycle length under laboratory conditions to be 3 and 4 days. From these data, we calculated the Cx. quinquefasciatus survival rate to be 0.60 and 0.68 per day. Density of the Cx. quinquefasciatus female (5.71 females per m(2)) was estimated based on a population size of 28,810 individuals divided by the sampled area of 5,040 m(2). Results of all experiments indicate medium survivorship and high density of the Cx. quinquefasciatus female population. This species is epidemiologically relevant in the PET area and should be a target of the vector control program of Sao Paulo municipality.

LARVICIDAL ACTIVITY OF GRANDISIN AGAINST AEDES AEGYPTI

Dengue is a tropical disease caused by an arbovirus transmitted by the mosquito Aedes aegypti. Because no effective vaccine is available for the disease, the strategy for its prevention has focused on vector control by the use of natural insecticides. The aim of this study was to evaluate the larvicidal activity of the lignan grandisin, a leaf extract from Piper solmsianum, against Ae. aegypti.

Avaliação da atividade antimalárica de extratos obtidos de algas marinhas no litoral do Rio Grande do Norte

Malaria is a major parasitic disease worldwide, accounting for about 500 million cases and causing 2 million to 3 million deaths annually. Four species are responsible for transmitting this disease to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The parasite resistance to antimalarial drugs and the usual limitations of the vector control implications are contributing to the spread of the disease. The most of significant advances in the search for new antimalarial drugs is based on natural components, the main ones being currently used antimalarial drugs derived from plants. Research on natural products of marine origin (particularly algae) show that some species possess antiplasmodial activity. Knowing that the coast of Rio Grande do Norte is home to several species of algae, the present study was to evaluate, for the first time, the antimalarial activity of ethanolic extracts of seaweed Spatoglossum schroederi, Gracilaria birdiae and Udotea flabellum against Plasmodium falciparum 3D7 strain tests and in vitro using the murine model (Plasmodium berghei) for evaluation in vivo. These species were ground, macerated with ethanol for 24 hours and the extracts concentrated in rotaevaporador (45 ° C ± 5 ° C). For in vitro tests, the extracts were diluted and tested at concentrations between 100 and 1.56 μg/ml (seven concentrations in triplicate), in order to obtain IC50 of each extract. The cytotoxicity tests with macrophages and BGM were performed using the MTT colorimetric assay. BGM macrophages and cells were distributed in 96 wells per plate (1x 105 to macrophages and 1x104 cells per well for BGM) and incubated for 24h at 37 ° C. The ethanol extracts were diluted and tested at concentrations of 100 to 1,56 μg/ml (seven concentrations in triplicate). After periods of 24 hours of incubation with the extracts, 100 μg of MTT was added to each well, and 3 hours elapsed, the supernatant was removed and added 200 μl of DMSO in each well. The absorbance of each well was obtained by reading on a spectrophotometer at 570 nm filter. To evaluate the acute toxicity in vivo, Swiss mice received a single dose (oral) 2000 mg/kg/animal of each extract tested. The parameters of acute toxicity were observed for 8 days. For in vivo tests, Swiss mice were inoculated with 1x105 erythrocytes infected with P. berghei. The treatment was given first to fourth day after infection with 0.2 ml of the extracts in doses of 1000 and 500 mg//g animal. The negative control group received 0.2 ml of 2% Tween-20, whereas the positive control group received sub-dose of chloroquine (5 mg/kg/animal). The assessment of antimalarial activity was done by suppressing suppressing the parasitemia at 5 and 7 days after infection. The growth inhibition of parasites was determined relative to negative control (% inhibition = parasitaemia in control - parasitemia in sample / parasitemia control x 100), the mortality of animals was monitored daily for 30 days The results showed that algae Spatoglossum schroederi and Udotea flabellum showed antimalarial activity in vitro, with reduced parasitemia of 70.54% and 54, respectively. The extracts of the three algae tested showed moderate to high cytotoxicity. Algae S. schroederi and U. flabellum were active against P. berghei only at doses of 500 mg / kg with reduction ranging from 54.58 to 52.65% for the fifth day and from 32.24 to 47.34% for the seventh day, respectively. No toxicity was observed in vivo at the dose tested, over the 8 days of observation. Although preliminary data, the bioactive components in those possible seaweed may be promising for the development of new anti-malarial drugs

Controle vetorial de velocidade de uma máquina de indução sem mancais trifásica com bobinado dividido utilizando estimação neural de fluxo

This work describes the study and the implementation of the vector speed control for a three-phase Bearingless induction machine with divided winding of 4 poles and 1,1 kW using the neural rotor flux estimation. The vector speed control operates together with the radial positioning controllers and with the winding currents controllers of the stator phases. For the radial positioning, the forces controlled by the internal machine magnetic fields are used. For the radial forces optimization , a special rotor winding with independent circuits which allows a low rotational torque influence was used. The neural flux estimation applied to the vector speed controls has the objective of compensating the parameter dependences of the conventional estimators in relation to the parameter machine s variations due to the temperature increases or due to the rotor magnetic saturation. The implemented control system allows a direct comparison between the respective responses of the speed and radial positioning controllers to the machine oriented by the neural rotor flux estimator in relation to the conventional flux estimator. All the system control is executed by a program developed in the ANSI C language. The DSP resources used by the system are: the Analog/Digital channels converters, the PWM outputs and the parallel and RS-232 serial interfaces, which are responsible, respectively, by the DSP programming and the data capture through the supervisory system

Controle vetorial de velocidade de um motor de indução trifásico com estimação neural de fluxo

This work describes the study and the implementation of the speed control for a three-phase induction motor of 1,1 kW and 4 poles using the neural rotor flux estimation. The vector speed control operates together with the winding currents controller of the stator phasis. The neural flux estimation applied to the vector speed controls has the objective of compensating the parameter dependences of the conventional estimators in relation to the parameter machine s variations due to the temperature increases or due to the rotor magnetic saturation. The implemented control system allows a direct comparison between the respective responses of the speed controls to the machine oriented by the neural rotor flux estimator in relation to the conventional flux estimator. All the system control is executed by a program developed in the ANSI C language. The main DSP recources used by the system are, respectively, the Analog/Digital channels converters, the PWM outputs and the parallel and RS-232 serial interfaces, which are responsible, respectively, by the DSP programming and the data capture through the supervisory system

Controle vetorial para velocidade de um motor de indução trifásico utilizando estimador filtro de Kalman

This paper describes the study, computer simulation and feasibility of implementation of vector control speed of an induction motor using for this purpose the Extended Kalman Filter as an estimator of rotor flux. The motivation for such work is the use of a control system that requires no sensors on the machine shaft, thus providing a considerable cost reduction of drives and their maintenance, increased reliability, robustness and noise immunity as compared to control systems with conventional sensors

Genetic differences between strains of Biomphalaria glabrata (Planorbidae) that are susceptible and unsusceptible to schistosomiasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoramento da suscetibilidade de populações de Triatoma sordida Stål, 1859 (Hemiptera: Reduviidae) ao inseticida deltametrina, na região Centro-Oeste do Brasil

INTRODUÇÃO: Populações de Triatoma sordida Stål, 1859 foram investigadas quanto à suscetibilidade à deltametrina. MÉTODOS: Análise por meio de bioensaios por aplicação tópica em 11 populações de T. sordida procedentes dos Estados de Goiás, Mato Grosso e Mato Grosso do Sul. RESULTADOS: As estimativas de DL50 e RR50 demonstraram elevados níveis de suscetibilidade (DL50 < 1 e RR50 < 2). Entretanto, as análises do coeficiente angular da curva dose resposta revelaram que as populações de triatomíneos dos municípios de Firminópolis/GO, Posse/GO, Poxoréu/MT, Douradina/MS e Aparecida do Taboado/MS apresentam maiores probabilidades de evolução de resistência, portanto, mais propícias a tolerar o tratamento com deltametrina. CONCLUSÕES: Detectaram-se pequenas alterações de suscetibilidade e baixos níveis de resistência, porém as alterações temporais de suscetibilidade deverão ser continuamente monitoradas, a fim de nortear adequadamente as ações de controle dos vetores da DC.

A novel HPF voltage source rectifier for variable speed refrigeration systems

A novel single-phase voltage source rectifier capable to achieve High-Power-Factor (HPF) for variable speed refrigeration system application, is proposed in this paper. The proposed system is composed by a single-phase high-power-factor boost rectifier, with two cells in interleave connection, operating in critical conduction mode, and employing a soft-switching technique, controlled by a Field Programmable Gate Array (FPGA), associated with a conventional three-phase IGBT bridge inverter (VSI - Voltage Source Inverter), controlled by a Digital Signal Processor (DSP). The soft-switching technique for the input stage is based on zero-current-switching (ZCS) cells. The rectifier's features include the reduction in the input current ripple, the reduction in the output voltage ripple, the use of low stress devices, low volume for the EMI input filter, high input power factor (PF), and low total harmonic distortion (THD) in the input current, in compliance with the EEC61000-3-2 standards. The digital controller for the output stage has been developed using a conventional voltage-frequency control (scalar V/f control), and a simplified stator oriented Vector control, in order to verify the feasibility and performance of the proposed digital controls for continuous temperature control applied at a refrigerator prototype.

Association of insecticide use and alteration on Aedes aegypti susceptibility status

Dengue and dengue hemorrhagic fever, vector-borne diseases transmitted by the mosquito Aedes aegypti, are presently important public health problems in Brazil. As the strategy for disease control is based on vector control through the use of insecticides, the development of resistance is a threat to programs efficacy. The objective of this study was to compare the Aedes aegypti susceptibility in nine vector populations from the state of São Paulo and seven from Northeast region of Brazil, since there was a difference on group of insecticide used between the areas. Bioassays with larvae and adult were performed according to the World Health Organization methods.The results showed higher resistance levels to organophosphates group in populations from the Northeast region where this group was used for both larvae and adult control than in São Paulo where organophosphates were used for larvae and pyretroids for adult control. Resistance to pyretroids in adults was widespread in São Paulo after ten years of use of cypermethrin while in vector populations from the Northeast region it was punctual. The difference in resistance profile between the areas is in accordance to the group of insecticide used.

Dog culling and replacement in an area endemic for visceral leishmaniasis in Brazil

Measures employed to control visceral leishmaniasis in Brazil have focused on vector control by residual insecticide spraying and diagnosis of infection with elimination of positive dogs. We describe dog culling and replacement in a Brazilian endemic area (the Alvorada District, Aracatuba, SP) in order to better understand dog population dynamics when elimination of the dog reservoir is adopted as the main control measure. From August 2002 to July 2004, 60.9% of the estimated dog population for the area was culled with a mean age of 34 months old. The presence of anti-Leishmania sp. antibodies was recorded for only 26.7% of the euthanized canines. Replacement was observed in 38.8% of the cases, some of them by 2 or more dogs and in a mean time of 4 months. Dogs were replaced mostly by puppies of both sexes with a mean age of 6.8 months. From August 2002 to April 2005 we were able to follow-up 116 of these dogs, during a mean time of 8.7 months. Canine visceral leishmaniasis seropositivity by ELISA was observed in 42.2% of the followed dogs, 30.6% of which were already positive at the first evaluation. By the end of the follow-up period 37% of the dogs were submitted to euthanasia, with a mean age of 18.3 months. In the studied CVL endemic area of Brazil, euthanasia and the subsequent replacement ratio were high, increasing the dog population turnover and leading to a younger population that might be more susceptible to a variety of other infectious diseases in addition to CVL. Dog culling as a control strategy for VL should be reassessed. (C) 2008 Elsevier B.V. All rights reserved.

Standardization of bioassays for monitoring resistance to insecticides in Aedes aegypti

Since the reintroduction of Aedes aegypti in Brazil in the 1980s, insecticide use for its control is routine. The chemical control efficacy is threatened by vectors developing resistance to insecticides. The World Health Organization, recognizing the impact of insecticide resistance in vector control programmes, proposed standardizing bioassays for detecting and monitoring resistance using a diagnostic dose method. As Brazil has a national programme for monitoring the resistance of Ae. aegypti populations to insecticides, this study was designed to compare diagnostic bioassays at WHO suggested concentrations and those estimated for local conditions. Populations were resistant to both temephos doses. But important differences were seen for fenitrothion and malathion, which could lead to under- or over-estimation of resistance respectively. These results and inclusion of a diagnostic dose bioassay standard for larvae are discussed.

Variable speed refrigeration system with HPF input rectifier stage

This paper is based on the analysis and implementation of a new drive system applied to refrigeration systems, complying with the restrictions imposed by the IEC standards (Harmonic/Flicker/EMI-Electromagnetic Interference restrictions), in order to obtain high efficiency, high power factor, reduced harmonic distortion in the input current and reduced electromagnetic interference, with excellent performance in temperature control of a refrigeration prototype system (automatic control, precision and high dynamic response). The proposal is replace the single-phase motor by a three-phase motor, in the conventional refrigeration system. In this way, a proper control technique can be applied, using a closed-loop (feedback control), that will allow an accurate adjustment of the desirable temperature. The proposed refrigeration prototype uses a 0.5Hp three-phase motor and an open (Belt-Drive) Bitzer IY type compressor. The input rectifier stage's features include the reduction in the input current ripple, the reduction in the output voltage ripple, the use of low stress devices, low volume for the EMI input filter, high input power factor (PF), and low total harmonic distortion (THD) in the input current, in compliance with the IEC61000-3-2 standards. The digital controller for the output three-phase inverter stage has been developed using a conventional voltage-frequency control (scalar V/f control), and a simplified stator oriented Vector control, in order to verify the feasibility and performance of the proposed digital controls for continuous temperature control applied at the refrigerator prototype. ©2008 IEEE.

Biologic and genetics aspects of chagas disease at endemic areas

The etiologic agent of Chagas Disease is the Trypanosoma cruzi, transmitted through blood-sucking insect vectors of the Triatominae subfamily, representing one of the most serious public health concerns in Latin America. There are geographic variations in the prevalence of clinical forms and morbidity of Chagas disease, likely due to genetic variation of the T. cruzi and the host genetic and environmental features. Increasing evidence has supported that inflammatory cytokines and chemokines are responsible for the generation of the inflammatory infiltrate and tissue damage. Moreover, genetic polymorphisms, protein expression levels, and genomic imbalances are associated with disease progression. This paper discusses these key aspects. Large surveys were carried out in Brazil and served as baseline for definition of the control measures adopted. However, Chagas disease is still active, and aspects such as host-parasite interactions, genetic mechanisms of cellular interaction, genetic variability, and tropism need further investigations in the attempt to eradicate the disease. Copyright 2012 Marilanda Ferreira Bellini et al.