Evaluation of Antiviral Activity of Cyclic Ketones against Mayaro Virus.

Mayaro virus (MAYV) is a neglected arthropod-borne virus found in the Americas. MAYV infection results in Mayaro fever, a non-lethal debilitating disease characterized by a strong inflammatory response affecting the joints and muscles. MAYV was once considered endemic to forested areas in Brazil but has managed to adapt and spread to urban regions using new vectors, such as Aedes aegypti, and has the potential to cause serious epidemics in the future. Currently, there are no vaccines or specific treatments against MAYV. In this study, the antiviral activity of a series of synthetic cyclic ketones were evaluated for the first time against MAYV. Twenty-four compounds were screened in a cell viability assay, and eight were selected for further evaluation. Effective concentration (EC50) and selectivity index (SI) were calculated and compound 9-(5-(4-chlorophenyl]furan-2-yl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2))-dione (9) (EC50 = 21.5 µmol·L-1, SI = 15.8) was selected for mechanism of action assays. The substance was able to reduce viral activity by approximately 70% in both pre-treatment and post-treatment assays.

DENV-3 precursor membrane (prM) glycoprotein enhances E protein immunogenicity and confers protection against DENV-2 infections in a murine model.

To improve a DNA vaccine containing the truncated dengue virus serotype 2 (DENV-2) envelope (E) protein and evaluate the influence of precursor membrane (prM) glycoprotein polymorphism on E protein immunogenicity, two vaccine candidates have been constructed by upstream insertion of the DENV-2 and DENV-3 prM genes into the DENV-2 E gene, named pCID2EtD2prM and pCID2EtD3prM, respectively. Both constructs were able to induce antibody production, which were neutralizing against DENV-2 in a murine model. Splenocytes of immunized groups, when challenged with virus, demonstrated Th1 cytokine pattern and proliferation, in addition to the increase of specific T cells. Vaccine candidates pCID2EtD2prM and pCID2EtD3prM confer 70% and 90% protection against DENV-2, respectively. The pCID2EtD3prM plasmid conferred only 40% protection in the lethal challenge with DENV-2. The results demonstrate that DENV-3 prM has a greater influence on the immunogenicity of the E protein and, probably due to its role as a chaperone, these results may be related to the correct folding and, consequently, an increase in the presentation efficiency of produced transcripts.

The plant organs and rhizosphere determine the common bean mycobiome.

The plant microbiota diversity is often underestimated when approaches developed mainly for the identification of cultivable microorganisms are used. High-throughput sequencing allows a deeper understanding of the microbial diversity associated with plants. The amplification of ITS1 was used to analyze fungal diversity in several plant organs and rhizosphere of three common bean (Phaseolus vulgaris) varieties grown in a greenhouse. The fungal diversity diverged between those plant organs and the rhizosphere, with the highest found in the rhizosphere and the lowest in the stem. In each organ different numbers of genus, OTUs were identified, in a total of 283 OTUs evenly distributed among the varieties. In the co-occurrence network, a larger number of positive interactions were found in the organs of the aerial part in all varieties. We observed that the diversity of the endophytic microbiota differed more between plant organs than between common bean varieties. Our results show that the diversity of endophytic fungi can be efficiently accessed with the sequencing of ITS amplicons and that this diversity may vary among distinct plant organs and the rhizosphere of a single plant variety.

Discovery of novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds.

The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 µmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease.

Integrated diversity analysis of the microbial community in a reverse osmosis system from a Brazilian oil refinery.

Oil refineries are known for the large volume of water used in their processes, as well as the amount of wastewater generated at the end of the production chain. Due to strict environmental regulations, the recycling of water has now become a viable alternative for refineries. Among the many methods available to treat wastewater for reuse, the use of membranes in reverse osmosis systems stands out due to several economic and environmental benefits. However, these systems are vulnerable to contamination and deposition of microorganisms, mainly because of the feedwater quality. In this study, the microbial diversity of feedwater and reverse osmosis membranes was investigated using a combination of culture-dependent and culture-independent methods in order to characterize the microorganisms colonizing and deteriorating the membranes. In total, 37 bacterial isolates, 17 filamentous fungi and approximately 400 clones were obtained and analyzed. Among the bacterial genera identified, the most represented were Sphingobium, Acidovorax, Microbacterium, Rhizobium and Shinella. The results revealed genera that acted as candidate key players in initial biofilm formation in membrane systems, and provided important information concerning the microbial ecology of oligotrophic aquatic systems.

The antimicrobial peptide HS-1 inhibits dengue virus infection.

Dengue virus (DENV) is an arbovirus that belongs to the Flaviviridae family. Studies reveal that peptides secreted by amphibians have many functions, such as antiviral and antimicrobial activities. As there is no antiviral drug effective against the DENV, the antiviral activity of a synthetic peptide called HS-1, derived from the secretion of the anuran Hypsiboas semilineatus, has been evaluated. The assays of neutralization in the Vero cells show a complete inhibition of infection of the serotypes 2 and 3. Furthermore, the direct action of peptides on the viral particle can be observed through atomic force microscopy. In vivo tests display 80% protection against the dengue-2 virus due to the presence of HS-1, which reveals its potential as an antiviral against the DENV.

Functional and genetic characterization of hydrocarbon biodegrader and exopolymer-producing clones from a petroleum reservoir metagenomic library.

Microbial degradation of petroleum is a worldwide issue, which causes physico-chemical changes in its compounds, diminishing its commercial value. Biosurfactants are chemically diverse molecules that can be produced by several microorganisms and can enable microbial access to hydrocarbons. In order to investigate both microbial activities, function-driven screening assays for biosurfactant production and hydrocarbon biodegradation were carried out from a metagenomic fosmid library. It was constructed from the total DNA extracted from aerobic and anaerobic enrichments from a Brazilian biodegraded petroleum sample. A sum of 10 clones were selected in order to evaluate their ability to produce exopolymers (EPS) with emulsifying activity, as well as to characterize the gene sequences, harbored by the fosmid clones, through 454 pyrosequencing. Functional analyses confirmed the ability of some clones to produce surfactant compounds. Regarding hydrocarbon as microbial carbon sources, n-alkane (in mixture or not) and naphthalene were preferentially consumed as substrates. Analysis of sequence data set revealed the presence of genes related to xenobiotics biodegradation and carbohydrate metabolism. These data were corroborated by the results of hydrocarbon biodegradation and biosurfactant production detected in the evaluated clones.

Complete Genome Sequence of vB_EcoM-UFV13, a New Bacteriophage Able To Disrupt Trueperella pyogenes Biofilm.

vB_EcoM-UFV13, a member of the T4virus genus, shows lytic activity against Escherichia coli and effectiveness in controlling the biofilm formed by Trueperella pyogenes, which qualifies it as a promising component of phage cocktails for mastitis and metritis control.

Multi-walled carbon nanotubes increase antibody-producing B cells in mice immunized with a tetravalent vaccine candidate for dengue virus.

BACKGROUND: In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile. RESULTS: Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC. CONCLUSIONS: The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

UFV-P2 as a member of the Luz24likevirus genus: a new overview on comparative functional genome analyses of the LUZ24-like phages.

BACKGROUND: Phages infecting spoilage microorganisms have been considered as alternative biocontrol agents, and the study of their genomes is essential to their safe use in foods. UFV-P2 is a new Pseudomonas fluorescens-specific phage that has been tested for its ability to inhibit milk proteolysis. RESULTS: The genome of the phage UFV-P2 is composed of bidirectional modules and presented 75 functionally predict ORFs, forming clusters of early and late transcription. Further genomic comparisons of Pseudomonas-specific phages showed that these viruses could be classified according to conserved segments that appear be free from genome rearrangements, called locally collinear blocks (LCBs). In addition, the genome organization of the phage UFV-P2 was shown to be similar to that of phages PaP3 and LUZ24 which have recently been classified as a Luz24likevirus. CONCLUSIONS: We have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.

Dengue-1 envelope protein domain III produced in Pichia pastoris: potential use for serological diagnosis.

Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.

Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

Complete Genome Sequence of the Pseudomonas fluorescens Bacteriophage UFV-P2.

Milk proteolysis caused by Pseudomonas fluorescens is a serious problem in the dairy industries as a result of its ability to grow under refrigeration. The use of phages to control contaminants in food has been considered an alternative to traditional methods; therefore, a thorough understanding of such organisms is vital for their use. In this study, we show the complete genome sequence and analysis of a P. fluorescens phage isolated from wastewater of a dairy industry in Brazil.

Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system.

In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation.

Investigation of bacterial diversity in membrane bioreactor and conventional activated sludge processes from petroleum refineries using phylogenetic and statistical approaches.

Bacterial diversity of two distinct wastewater treatment systems, conventional activated sludge (CAS) and membrane bioreactor (MBR), of petroleum refineries were investigated through 16S rRNA gene libraries. Sequencing and phylogenetic analysis showed that the bacterial community composition of sludge samples was distinct between the two wastewater treatment systems. MBR clones belonged predominantly to Class Betaproteobacteria, represented mainly by genera Thiobacillus and Thauera, whereas CAS clones were mostly related to Class Alphaproteobacteria, represented by uncultured bacteria related to Order Parvularculales. Richness estimators ACE and Chao revealed that the diversity observed in both libraries at the species level is an underestimate of the total bacterial diversity present in the environment and further sampling would yield an increased observed diversity. Shannon and Simpson diversity indices were different between the libraries and revealed greater bacterial diversity for the MBR library, considering an evolutionary distance of 0.03. LIBSHUFF analyses revealed that MBR and CAS communities were significantly different at the 95% confidence level (P< or =0.05) for distances 0< or =D< or =0.20. This work described, qualitatively and quantitatively, the structure of bacterial communities in industrial-scale MBR and CAS processes of the wastewater treatment system from petroleum refineries and demonstrated clearly differentiated communities responsible for the stable performance of wastewater treatment plants.

Monitoring the bacterial community dynamics in a petroleum refinery wastewater membrane bioreactor fed with a high phenolic load.

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.