RESUMO

The limited availability of antivirals for new highly pathogenic strains of virus has become a serious public health. Therefore, news products against these pathogens has become an urgent necessity. Among the multiple sources for news antibiotics and antivirals, insect exudates or their products has become an increasingly frequent option. Insects emerged 350 million years ago and have showed a high adaptability and resistance to the most varied biomes. Their survival for so long, in such different environments, is an indication that they have a very efficient protection against environmental infections, despite not having a developed immune system like mammals. Since the ancient civilizations, the products obtained from the bee have been of great pharmacological importance, being used as antimicrobial, anti-inflammatory, antitumor and several other functions. Investigations of biological activity of propolis have been carried out, mainly in the species Apis mellifera, and its product have showed activity against some important viruses. However, for the Meliponini species, known as stingless bees, there are few studies, either on their chemical composition or on their biological activities. The importance of studying these bees is because they come from regions with native forests, and therefore with many species of plants not yet studied, in addition to which they are regions still free of pesticides, which guarantees a greater fidelity of the obtained data. Previous studies by our group with crude hydroalcoholic extract of propolis demonstrated an intense antiviral activity against Herpes, influenza, and rubella viruses. In this work, we chose to use aqueous extracts, which eliminates the presence of other compounds besides those originally present in propolis, in addition to extracting substances different from those obtained in alcoholic extracts. Therefore, this study aimed to identify, isolate and characterize compounds with antiviral effects from aqueous propolis extracts from Scaptotrigona aff postica, in emerging viruses such as zicavirus, chikungunya, and mayaro virus. The evaluation of the antiviral activity of the crude and purified material was performed by reducing infectious foci in VERO cell cultures. The results obtained with crude propolis, indicate a high reduction of zica virus (64×) and mayaro (128×) when was used 10% v/v of propolis. The reduction of chikungunya virus was of 256 fold, even when was used 5% v/v of propolis. The chemical characterization of the compounds present in the extracts was performed by high-pressure liquid chromatography. Through the purification of propolis by HPLC and mass spectrometry, it was possible to identify and isolate a peak with antiviral activity. This substance showed activity against all viruses tested. When purified fraction was used, the reduction observed was of 16 fold for zicavirus, 32 fold for mayaro virus and 512 fold for chikungunya virus. Likewise, it was observed that the antiviral response was concentration dependent, being more intense when propolis was added 2 h after the viral infection. Now we are carrying out the chemical characterization of the purified compounds that showed antiviral action.

Assuntos

RESUMO

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.

RESUMO

The use of natural products has a long tradition in medicine, and they have proven to be an important source of lead compounds in the development of new drugs. Among the natural compounds, terpenoids present broad-spectrum activity against infective agents such as viruses, bacteria, fungi, protozoan and helminth parasites. In this study, we report a biological screening of 38 chemically characterized terpenes from different classes, which have a hydroxyl group connected by hydrophobic chain or an acceptor site, against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. In vitro bioassays revealed that 3,7-dimethyl-1-octanol (dihydrocitronellol) (10) was the most active terpene (IC50 values of 13­52 µM) and, thus, we investigated its antischistosomal activity in greater detail. Confocal laser scanning microscopy revealed that compound 10 induced severe tegumental damage in adult schistosomes and a correlation between viability and tegumental changes was observed. Furthermore, we compared all the inactive compounds with dihydrocitronellol structurally by using shape and charge modeling. Lipophilicity (miLogP) and other molecular properties (e.g. molecular polar surface area, molecular electrostatic potential) were also calculated. From the 38 terpenes studied, compound 10 is the one with the greatest flexibility, with a sufficient apolar region by which it may interact in a hydrophobic active site. In conclusion, the integration of biological and chemical analysis indicates the potential of the terpene dihydrocitronellol as an antiparasitic agent.

Assuntos

RESUMO

The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

Assuntos

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In this study, we investigated in groups of female BALB/c mice injected with Crotalus durissus terrificus venom (Cdt) the renal function based on creatinine clearance, percentage of fractional excretion cytokines and histological examination of renal tissue. Cdt caused renal alterations that induced proteinuria during the initial hours post-venom and reduced creatinine clearance 15 min. up to 2 hours post-venom administration. In urine from mice injected with Cdt induced a decrease in IL-4 levels. More pronounced increments of IL-5, IL-6 and IFN-γ were observed after 15 and 30 min, respectively. The highest levels of TNF and IL-10 were observed at 1 and 4 hs, respectively. The ratios of pro- and anti-inflammatory cytokines in animals injected with Cdt, which may be manifested in the inflammatory status during the envenoming. In groups of animals treated with Cdt were observed a decreasing in creatinine clearance and its effect on glomerular filtration rate was accompanied by decreased fractional excretion of cytokines and morphologic disturbances. This loss of change selectively in envenomation could thus explain why the relatively excretion of cytokines is reduced while of total proteins increases. In conclusion the fractional excretion of cytokines is significantly reduced in mice injected with Cdt, despite proteinuria.

Assuntos

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Levels of antibody against influenza virus were evaluated in serum pairing samples from individuals immunized against influenza by Hemagglutination Inhibition and Single Radial Hemolysis tests. For this purpose, groups of smokers, non-smokers and, of those holding respiratory complications, were formed. Results of serologic titrations pointed out to an increase in the level of antibodies for the smoker and non-smoker groups, with significant degrees of difference up to P < 0.001 difference between both averages after immunization. However, in the group of respiratory complications no significant differences (P > 0.05) were found out between the averages antibody levels for the subtype A (H1N1) and the Type B (vaccine components); an increase only at the level of antibodies was registered, with differences among the averages of the antibody levels, for the subtype A (H3N2) (vaccine component) at degrees of P < 0.01 and P < 0.001 on the titration of the SRH and HI tests, respectively. It can demonstrate that immunization against influenza presents a good protection for the smoker and non-smoker groups; however, in the group of respiratory complications it only occurred with the subtype A (H3N2), indicating that this subtype presents good antigenicity since it has induced better formation of antibodies, even in defective organisms.

Assuntos

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The age distribution of antibody to simian rotavirus (SA-11) was studied in serum specimens obtained from 399 children aged to 5 years and living in the city of Recife (PE), located in the north eastern region of Brazil. Sera were examined for group-specific rotavirus antibody using a blocking enzyme immunoassay (bELISA) and a hemagglutination inhibition antibody (HIA) test, and for anti-VP2, anti-VP4, anti-VP6, and anti-VP7 antibodies using an immunoblotting assay (IBA). Antibody prevalence was similar in all bELISA and HIA assays, showing a steep rise in the 6-to 17-month-old age groups. The results indicate early acquisition of antibody to rotavirus. The majority of children aged 2 to 4 years had bELISA (50% to 60%) and HIA (70% to 81%) antibodies. There was an association in prevalence data obtained by HIA and bELISA with immunoblotting (IBA), revealing four serologic profiles. Children with profiles I and II (60%) respectively had HAI and ELISA antibody or HAI antibody alone and all had immunoprotective antibodies to VP4 and/or VP7. These children were regarded as "immune," resembling convalescent patients with a rotavirus infection. Children with profile III (4%) had no HIA antibody and only non-protective anti-VP6 and/or VP7 antibody, and were considered to be "partially immune." Children with profile IV (36%) had no detectable antibody and were classified as "nonimmune." These children should be considered to be susceptible to rotavirus infection, with the risk of developing clinically severe diarrhea.

Assuntos

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1. A large amount of antigen is required to conduct seroepidemiologic surveys of measles. Thus, a process to obtain measles virus antigen using a bioreactor was standardized. 2. The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm. The cultures infected with 0.5 m.o.i. of measles virus were harvested after the appearance of the cytopathic effect. The virus suspension was clarified and concentrated by ultracentrifugation. Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50). 3. Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg. In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg. 4. The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT). 5. The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.

Assuntos

RESUMO

1. A large amount of antigen is required to conduct seroepidemiologic surveys of measles. Thus, a process to obtain measles virus antigen using a bioreactor was standardized. 2. The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm. The cultures infected with 0.5 m.o.i. of measles virus were harvested after the appearance of the cytopathic effect. The virus suspension was clarified and concentrated by ultracentrifugation. Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50). 3. Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg. In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg. 4. The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT). 5. The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.

Assuntos

RESUMO

Genetically homogeneous and heterogeneous mouse populations were tested for resistance to experimental street rabies virus infection and their ability to synthesize interferon (IFN) during the infection. The genetically heterogenous HI mouse population was highly resistant (12% mortality), and the genetically homogeneous BALB/c and C3H mice as well as the genetically heterogeneous Sw and LI mouse populations were susceptible (60 to 71% mortality). The genetically homogeneous A/J mice were highly susceptible (85% mortality) to experimental street rabies infection. The ability of these mice to synthesize IFN as measured in serum 4 days after the infection was directly related to the degree of resistance, with the highly resistant HI mice showing large amounts of IFN (850 U/ml), and the susceptible mice showing low amounts of IFN (50 to 280 U/ml). IFN induced within the central nervous system and measured in brain homogenates during infection was not correlated with resistance. The present data suggest that high levels of IFN occurring in serum early during infection with street rabies virus contribute to the resistance of these mice.

Assuntos