José Carlos de Magalhães

Identification of a phylogenetically distinct orthobunyavirus from group C.

Apeu virus (APEUV) (family Bunyaviridae, genus Orthobunyavirus) was plaque purified and characterised by serological and molecular analysis. Neutralising assays confirmed cross-reactivity between purified APEUV clones and the Caraparu virus complex of group C orthobunyaviruses. Partial sequencing of the L, M and S segments of one APEUV clone (APEUV-CL5) was carried out. A phylogenetic tree constructed with the L amino acid sequences clustered APEUV-CL5 within the genus Orthobunyavirus, confirming its serological classification. Analysis of M segment sequences clustered APEUV-CL5 in the Caraparu virus complex (Group C), in agreement with serological tests and previous molecular characterisation. However, the sequence of the nucleocapsid gene (N) gave low identity values when compared to those of the group C viruses. The phylogenetic tree based on N nucleotide sequences clustered APEUV-CL5 next to the California and Bwamba groups. This remarkable S nucleotide variability suggests that APEUV-CL5 could be a genetic reassortant and that this evolutionary mechanism is present in the history of the group C viruses.

Dengue virus detection in Aedes aegypti larvae from southeastern Brazil

ABSTRACT: The transmission of dengue, the most important arthropod-borne viral disease in Brazil, has been intensified over the past decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index (EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four Ae. aegypti pools.

Dengue outbreaks in Divinopolis, south‐eastern Brazil and the geographic and climatic distribution of Aedes albopictus and Aedes aegypti in 2011–2012

To entomologically monitor Aedes spp. and correlate the presence of these vectors with the recent epidemic of dengue in Divinopolis, Minas Gerais State, Brazil.

Oxidative stress in Mayaro virus infection

Mayaro virus (MAYV) is a neglected tropical arbovirus that causes a febrile syndrome that is sometimes accompanied by incapacitating arthritis/arthralgia. The pathogenesis of MAYV has not been completely defined and oxidative stress mediated by an increase in reactive oxygen species (ROS) and/or depletion of antioxidant defences has been found to contribute to several aspects of viral disease. To investigate whether MAYV induced oxidative stress in host cells, we monitored ROS production, oxidative stress markers and antioxidant defences at different time points after infection. Our results show that MAYV induced significant oxidative stress in infected HepG2 cells, as indicated by the increase of malondialdehyde (MDA) and protein carbonyl levels, and by a significant decrease of the reduced versus oxidized glutathione (GSH/GSSG) ratio. Generally, MAYV-infected HepG2 cells also showed an increase in antioxidant defences. We observed an increase in the superoxide dismutase (SOD) and catalase (CAT) activities and the total glutathione content. To determine whether similar effects occurred in other cell types, we evaluated the ROS, MDA and SOD activity levels in J774 cells after MAYV infection. Similar to our observations in HepG2 cells, the J774 cells showed an increase in ROS, MDA and total SOD activity following MAYV infection. Thus, since the cellular redox environment is influenced by the production and removal of ROS, we hypothesize that the overproduction of ROS was responsible for the oxidative stress in response to the MAYV infection despite the increase in the antioxidant status. This study is the first report on the involvement of oxidative stress during MAYV infection. Collectively, our data shed light on some mechanisms that are operational in host cells following exposure to MAYV.

Caraparu virus induces damage and alterations in antioxidant defenses in the liver of BALB/c mice after subcutaneous infection

Oxidative stress is a disturbance in the oxidant-antioxidant balance leading to potential cellular damage. Most cells can tolerate a mild degree of oxidative stress because they have a system that counteracts oxidation that includes antioxidant molecules such as glutathione (GSH) and superoxide dismutase (SOD). Disruption of the host antioxidant status has been recognized as an important contributor to the pathogenesis of many viruses. Caraparu virus (CARV) is a member of group C of the Bunyaviridae family of viruses. In South American countries, group C bunyaviruses are among the common agents of human febrile illness and have caused multiple notable outbreaks of human disease in recent decades; nevertheless, little is known about the pathogenic characteristics of these viruses. The purpose of this study was to examine the hepatic pathogenesis of CARV in mice and the involvement of oxidative stress and antioxidant defenses on this pathology. Following subcutaneous infection of BALB/c mice, CARV was detected in the liver, and histopathology revealed acute hepatitis. Increased serum levels of aspartate and alanine aminotransferases (AST/ALT) and greater hepatic expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) were found in infected animals. CARV infection did not alter the biomarkers of oxidative stress but caused an increase in GSH content and altered the expression and activity of SOD. This is the first report of an alteration of oxidative homeostasis upon CARV infection, which may, in part, explain the hepatic pathogenesis of this virus, as well as the pathogenesis of other Bunyaviridae members.

Antibacterial and cytotoxic antibacterial potential of ethanol extract and fractions from Aristolochia galeata Mart. ex Zucc

The aim of this investigation was to evaluate the phytochemical, antibacterial and cytotoxic activities of ethanol extract and hexane, dichlorometane, ethyl acetate and hydroethanolic fractions from Aristolochia galeata’s rhizomes. The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) were evaluated by the broth microdilution assay to investigate the antibacterial activity of various extracts and fractions of A. galeata against Gram-positive and Gram-negative bacteria. The cytotoxicity of plant samples was evaluated in human cervix carcinoma cell line (HeLa) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The phytochemical study showed the presence of main secondary metabolites, steroids, flavonoids, coumarins and alkaloids. The ethanol extract and their fractions presented antimicrobial activity against Gram-positive bacteria, and Staphylococcus aureus was regarded the most sensitive strain with MIC of 250 µg/ml for the ethanol extract. The dichlorometane fraction showed bactericidal activity with the value of 1250 µg/ml and moderate cytotoxicity in front of the HeLa cell line tested (CC50 = 90 µg/ml). The results showed that A. galeata had effective antibacterial activity against Gram-positive bacteria and compounds extracted from A. galeata Mart. ex Zucc could be used as possible antimicrobials. The good antimicrobial activity and the low cytotoxicity presented by the hexane fraction can be promised for the new molecules with antibiotic activity.

Insignificant level of in vitro cytotoxicity, anti-rotavirus, antibacterial, and antifungal activities of N-alkylmaleamic acids

By reacting maleic anhydride with amines, we synthesized the derivatives N-ethyl, N-(2-ethylamine), N-piperidinyl, N-phenyl, and N-phenylhydrazinyl maleamic acids. The purity of these products was initially verified by melting range and the presence of only one spot observed by thin layer chromatography. The chemical structures of the obtained N-alkyl maleamic acids were confirmed through infrared (IR) and hydrogen and carbon nuclear magnetic resonance ( 1 H and 13 C NMR) spectrometry. Due to the already proven pharmacological activity of maleimides, maleic anhydride and its N-alkyl maleamic acids were subjected to in vitro assays to observe antiviral (SA-11 rotavirus), antibacterial (Escherichia coli, Staphylococcus aureus, and Bacillus cereus), antifungal (Colletotrichum musae, Fusarium solani f. sp. phaseoli, Fusarium solani f. sp. piperis Alb., and Penicillium sp.), and antiprotozoal (Trichomonas vaginalis, Giardia lamblia, and Entamoeba histolytica) effects. To study the anti-rotavirus properties, firstly the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) method was used to establish the median cytotoxicity concentration (CC 50 ) of the compounds, using MA-104 cell line. Under the experimental conditions used, cytotoxic, anti-rotavirus, antibacterial, and antifungal properties were not observed for these compounds.

Zika Virus Activity of the Leaf and Branch Extracts of Tontelea micrantha and Its Hexane Extracts Phytochemical Study

The new triterpene friedelan-1,3,21-trione, the known compounds friedelan-3-one, 3β-friedelinol, 3,4-seco-friedelan-3-oic acid, 28-hydroxyfriedelan-3-one, friedelan-3-oxo28-al, friedelan-3,21-dione, 30-hydroxyfriedelan-3-one, a mixture of 30-hydroxyfriedelan3-one/21α-hydroxyfriedelan-3-one, 21β-hydroxyfriedelan-3-one, gutta-percha, squalene, and a mixture of palmitic/stearic/oleic acids were isolated from the hexane extracts of leaves and branches of T. micrantha. Their chemical structures were established by Fourier transform infrared spectroscopy (FTIR), gas chromatography (GC), 1D/2D nuclear magnetic resonance (NMR) and comparison with the literature data. All compounds were described for T. micrantha and the genus Tontelea for the first time. The branch and leaf extracts displayed anti-Zika virus activity at the lowest tested concentration of 15.6 μg mL, mainly virucidal effect, and presented no cytotoxicity to Vero cells. Furthermore, the ethyl acetate and methanolic leaf extracts demonstrated the best activities at the concentration of 31.2 and 15.6 μg mL at the viral adsorption and penetration stages, respectively. These results showed that these extracts may be promising candidates for the Zika virus treatment.

High prevalence of dengue antibodies and the arginine variant of the FcγRIIa polymorphism in asymptomatic individuals in a population of Minas Gerais State, Southeast Brazil

Dengue is the most prevalent arthropod-borne viral illness in humans worldwide. Single-nucleotide polymorphisms (SNPs) in genes involved in the immune response, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), IgG Fc receptor II-A (FcγRIIa), vitamin D receptor (VDR), and tumor necrosis factor alpha (TNF-α), were previously reported to be associated with susceptibility to dengue disease in different human populations. Therefore, due to the relevant association of host immune and genetic status with disease susceptibility/severity of dengue, this work aims to verify the frequency of anti-dengue virus antibodies and some dengue-associated risk SNPs in a population in Minas Gerais State, Southeast Brazil. A total of 1560 individuals were genotyped for polymorphisms in DC-SIGN (rs4804803), FcγRIIa (rs1801274), VDR (rs7975232), and TNF-α (rs1800629). The presence of anti-dengue antibodies (IgM and/or IgG) in these samples was also assayed. Anti-dengue antibodies were detected at an overall frequency of 16.86%, indicating a virus infection in asymptomatic individuals. The genotypic frequencies of all SNPs studied did not differ between the asymptomatic and control groups. Regarding the allelic frequencies of the four SNPs analyzed, a higher frequency was detected of the G allele of FcγRIIa/rs1801274 in the asymptomatic individuals when compared to that in the control group (p = 0.03). Therefore, the results showed a high prevalence of asymptomatic individuals in Minas Gerais State, with a potential association between the presence of the G allele of FcγRIIa/rs1801274 and protection against symptomatic disease.

Antiviral activity of silymarin against Mayaro virus and protective effect in virus-induced oxidative stress

Mayaro virus (MAYV) is a neglected arbovirus belonging to the family Togaviridae. Its infection leads to Mayaro fever, with clinical manifestations such as fever, myalgia, headache, rash, arthralgia, vomiting, and diarrhea. The most prominent complaint from infected person is the long-lasting arthritis/arthralgia. The treatment for Mayaro fever is mainly symptom-based and there are no vaccines or antiviral drugs currently available, thus, natural products with anti-MAYV activity may provide a potential alternative. Recent evidences suggest that oxidative stress plays an important role in MAYV infection and compounds capable of modulating oxidative stress could represent a novel therapeutic approach in modulating MAYV-associated oxidative cellular damage. Silymarin is a complex extracted of Silybum marianum, or milk thistle, and its major active compound is silybin, which has a remarkable biological effect. Its antioxidant and antiviral effects, including its antiviral activity against the Chikungunya virus (CHIKV), prompted us to think whether silymarin could also reduce the replication of the MAYV and restore the pro-oxidant/antioxidant balance in the context of MAYV infection, leading to reduced cellular oxidative stress. We assessed the antiviral activity and protective effect of silymarin against oxidative stress in MAYV-infected HepG2 cells. Cytopathic effect inhibition, viral replication, and plaque reduction assays were used to determine the anti-MAYV activity of silymarin. Additionally, we determined whether silymarin could reduce MAYV-induced oxidative cell damage. Briefly, silymarin exhibited potent antiviral activity against MAYV and reduced MAYV-induced ROS formation and levels of malondialdehyde (MDA) and carbonyl protein, which are biomarkers of oxidative stress. In conclusion, the ability of silymarin to inhibit MAYV replication and attenuate MAYV-induce oxidative stress warrants further investigation of this compound as a novel therapeutic approach to Mayaro fever disease.