Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.

Biofilm production by multiresistant Corynebacterium striatumassociated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatumbound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

Dinâmica populacional de ácaros fitófagos (Acari, Eriophyidae, Tenuipalpidae) em seis clones de seringueira no sul do Estado de Mato Grosso

Foi estudada a dinâmica populacional de Calacarus heveae Feres, 1992, Phyllocoptruta seringueirae Feres, 1998 (Eriophyidae) e Tenuipalpus heveae Baker, 1945 (Tenuipalpidae) em seis clones de seringueira (Hevea brasiliensis Muell. Arg.), com o intuito de conhecer a sazonalidade e as possíveis diferenças nos níveis de infestação dessas espécies sobre os diferentes clones, em seringal localizado no sul do Estado de Mato Grosso. Para isso, no período de março de 2004 a março de 2005, foram realizadas coletas num intervalo médio de catorze dias em seis parcelas clonais. Em cada amostragem, foram analisados 100 folíolos de cada parcela, registrando-se o número total de ácaros encontrados em ambas as faces. Os níveis de infestação das espécies variaram entre os diferentes clones. O clone RRIM 600 apresentou maior abundância de T. heveae, enquanto que PB 235 e PB 260 de P. seringueira e C. heveae, respectivamente. Por outro lado, esse último clone apresentou menor número de indivíduos de T. heveae e P. seringueirae, e PB 235 de C. heveae. O primeiro semestre foi o período crítico de infestação, porém a duração da ocorrência e o pico de abundância dos fitófagos variaram nos diferentes clones.

Biological cycle of Tenuipalpus heveae Baker (Acari, Tenuipalpidae) on leaflets of three rubber tree clones

Life cycle of Tenuipalpus heveae Baker (Acari, Tenuipalpidae) on leaflets from three rubber tree clones. The biological cycle of Tenuipalpus heveae Baker, 1945 (Tenuipalpidae), a potential rubber tree pest mite, was studied by the observation of individuals reared on leaflets of the clones GT 1, PB 235 and RRIM 600, in controlled environmental conditions. Three daily observations were done of 60 eggs on leaflets from each clone in order to verify the development of immature stages and the female oviposition. The fertility life table was constructed based in the collected data. Mites reared on PB 235 had faster rate of development, requiring less time in days, to double its population in number (TD), and had the highest values for egg production, female longevity, net reproductive rate (Ro), intrinsic rate of natural increase (r m) and finite rate of increase (λ). Lower reproductive values and the longest time necessary to reach adult stage were recorded for the mites on GT 1. In all studied clones, the deutonymphal phase had the highest viability, while the larval phase had the lowest, highlighted by the survivorship curve that indicated high mortality during this life stage. The clone PB 235 allowed the most suitable conditions for the development of T. heveae, followed by RRIM 600, while GT 1 was the less suitable substratum to rear this mite species.

Efeitos da adubação NPK na fertilidade do solo, nutrição e crescimento da seringueira

O trabalho teve por objetivo avaliar o efeito de diferentes combinações de doses de fertilizantes NPK, durante o período de formação do seringal, sobre as características químicas do solo, nutrição mineral e crescimento de árvores de seringueira [Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg.], clone RRIM 600. O experimento foi instalado em 1985 e realizado até 1993, no delineamento de blocos ao acaso, num esquema fatorial fracionado 1/2 (4 x 4 x 4), testando as doses anuais de 0, 40, 80 e 120 kg ha-1 de N, P2O5 e K2O, aplicadas na forma de uréia, superfosfato triplo e cloreto de potássio. Foi usado Latossolo Vermelho-Amarelo Podzólico distrófico A moderado de textura arenosa/média, localizado no município de Avaí, estado de São Paulo, no período entre dois e oito anos de idade das árvores. A avaliação do experimento foi feita por meio da análise de solo, de folha e medida do perímetro do caule. O fertilizante nitrogenado (uréia) foi o que causou maiores alterações com intensa acidificação do solo. Além de alterar diversos atributos do solo, a aplicação de uréia elevou as concentrações de N e reduziu as de K e S nas folhas. A fertilização fosfatada aumentou a disponibilidade de P no solo e a concentração nas folhas, o mesmo acontecendo com a adubação potássica em relação à disponibilidade de K no solo e na planta. Apesar disso, as correlações entre as concentrações de P e K no solo e nas folhas e o período de imaturidade não foram coerentes com esse aumento de disponibilidade. Mesmo sem a aplicação de micronutrientes, observou-se que as concentrações de B nas folhas correlacionaram-se positivamente com o período de imaturidade, enquanto os demais micronutrientes pouco influíram na fertilidade do solo.

Cinética de absorção e eficiência nutricional de K+, Ca2+ e Mg2+ em plantas jovens de quatro clones de eucalipto

Modelos mecanísticos baseados em princípios de transporte de solutos pode ser de grande utilidade para prever os impactos do cultivo de florestas plantadas, por exemplo, com eucalipto sobre o capital e fluxo de nutrientes no solo. Dentre as variáveis de entrada demandadas por tais modelos, tem-se os valores das constantes Vmax, Km e Cmin da cinética de absorção iônica. Assim, os objetivos do presente trabalho foram determinar os valores de Vmax, Km e Cmin para K, Ca e Mg, bem como avaliar as respectivas eficiências nutricionais de clones de eucalipto. O estudo consistiu de três ensaios em solução nutritiva (um para cada cátion), sendo utilizadas mudas propagadas vegetativamente de um híbrido de E. grandis x E. urophylla (clone 1213) e de três híbridos de E. grandis (clones 7074, 57 e 129). Com base nos teores de cada um desses nutrientes nas soluções de depleção, em cada tempo de amostragem, no volume inicial e final de solução nos vasos e no peso de matéria fresca de raízes, foram obtidos os valores das constantes cinéticas. Para K, o clone 7074 apresentou o menor valor de Vmax em relação aos demais clones, os quais não diferiram entre si, e em relação ao Km e Cmin, os clones não diferiram estatisticamente. Para Ca, os clones estudados diferiram quanto ao valor de Vmax e Km, não diferindo, entretanto, para o Cmin. Os menores valores de Km para Mg foram verificados para os clones 57 e 7074, ou seja, as proteínas transportadoras de Mg na membrana plasmática das células radiculares apresentaram maior afinidade para esse nutriente. Contudo, os valores de Vmax e Cmin não diferiram entre os clones estudados. Diferenças na eficiência nutricional dos clones estudados quanto a K e a Ca foram devidas às diferenças na eficiência de absorção, e para Mg às diferenças na eficiência de absorção e de utilização.

Produção de matéria seca, crescimento radicular e absorção de cálcio, fósforo e alumínio por coffea canephora e coffea arabica sob influência da atividade do alumínio em solução

Este trabalho teve como objetivo avaliar a produção de matéria seca, o crescimento radicular e a absorção e distribuição do Ca, P e Al nas folhas, no caule e nas raízes de dois clones de café conilon (Coffea canephora) (Mtl 25 e Mtl 27) e de uma variedade de café Catuaí Amarelo (Coffea arabica), cultivados em solução nutritiva com atividade crescente de Al3+. As plantas foram cultivadas em vasos com capacidade para 5 L, contendo solução nutritiva de Hoagland & Arnon, modificada. Após oito dias de adaptação, as plantas foram submetidas a concentrações de Al de 0, 500, 1.000 e 2.000 µmol L-1, que corresponderam a atividades de Al3+ em solução, estimadas pelo software GEOCHEM, de 20,68, 50,59, 132,9 e 330,4 µmol L-1, respectivamente. Foram determinados os teores de Ca, Al e P na planta. O sistema radicular foi separado, para determinação da área e do comprimento. A variedade Catuaí Amarelo (Coffea arabica) apresentou-se menos sensível ao Al3+, quando comparada aos clones de conilon (Coffea canephora). O clone de conilon Mtl 25 foi menos sensível ao Al3+ em relação ao Mtl 27. O aumento da atividade de Al3+ promoveu redução nos teores de P e Ca nas folhas e raízes do cafeeiro, especialmente nos clones Mtl 25 e Mtl 27. O acúmulo de Al no sistema radicular e a restrição do transporte para a parte aérea são importantes fatores na tolerância de plantas ao Al3+.

Relação do estado nutricional de minicepas com o enraizamento de miniestacas de eucalipto

O enraizamento adventício de estacas é influenciado por fatores intrínsecos e extrínsecos do material vegetal, e o conhecimento da ação deles é fundamental para o sucesso da produção de mudas por miniestaquia. O objetivo deste trabalho foi avaliar o grau de associação entre o estado nutricional das minicepas e o enraizamento de miniestacas de eucalipto, cultivadas em minijardim clonal em tubetes com subirrigação. Foram utilizados dados de enraizamento de miniestacas e teores de nutrientes quantificados pelas análises químicas dos tecidos foliares. A taxa de enraizamento foi correlacionada com o estado nutricional das brotações. Verificou-se que a nutrição mineral desempenha papel importante no enraizamento adventício de modo genótipo-dependente, ou seja, há diferença de comportamento dos clones em relação ao efeito dos nutrientes no enraizamento das miniestacas. Com relação ao teor de K, Ca, Mg e Mn, não foi observada associação com as taxas de enraizamento. Quanto aos demais nutrientes, em pelo menos um clone, houve associação positiva ou negativa entre as variáveis analisadas, com grau variável de acordo com o clone e o nutriente.

Phloem mobility of Boron in two eucalypt clones

Boron deficiency causes large productivity losses in eucalypt stands in extensive areas of the Brazilian Cerrado region, thus understanding B mobility is a key step in selecting genetic materials that will better withstand B limitation. Thus, in this study B mobility was evaluated in two eucalypt clones (68 and 129), under B sufficiency or B deficiency, after foliar application of the 10B isotope tracer to a single mature leaf. Samples of young tissue, mature leaves and roots were collected 0, 1, 5, 12 and 17 days after 10B application. The 10B:11B isotope ratio was determined by HR-ICP-MS. Samples of leaves and xylem sap were collected for the determination of soluble sugars and polyalcohols by ion chromatography. Boron was translocated within eucalypt. Translocation of foliar-applied 10B to the young tissues, mature leaves and roots was higher in clone 129 than in 68. Seventeen days after 10B application to a single mature leaf, between 14 and 18 % of B in the young tissue was originated from foliar B application. In plants with adequate B supply the element was not translocated out of the labeled leaf.

Boron mobility in eucalyptus clones

Understanding the magnitude of B mobility in eucalyptus may help to select clones that are more efficient for B use and to design new practices of B fertilization. This study consisted of five experiments with three eucalyptus clones (129, 57 and 58) where the response to and mobility of B were evaluated. Results indicated that clone 129 was less sensitive to B deficiency than clones 68 and 57, apparently due to its ability to translocate B previously absorbed via root systems to younger tissues when B in solution became limiting. Translocation also occurred when B was applied as boric acid only once to a single mature leaf, resulting in higher B concentration in roots, stems and younger leaves. The growth of B-deficient plants was also recovere by a single foliar application of B to a mature leaf. This mobility was greater, when foliar-applied B was supplied in complexed (boric acid + manitol) than in non-complexed form (boric acid alone). When the root system of clone 129 was split in two solution compartments, B supplied to one root compartment was translocated to the shoot and back to the roots in the other compartment, improving the B status and growth. Thus, it appears that B is relatively mobile in eucalyptus, especially in clone 129, and its higher mobility could be due to the presence of an organic compound such as manitol, able to complex B.

Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation

Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI.

Effect of NPK fertilization on production and leaf nutrient content of eucalyptus minicuttings in nutrient solution

Adequate nutrient levels in plants vary according to the species or clone, age and management practice. Therefore, adjustments of the nutrient solution are often necessary according to the plant material for multiplication. This study aimed to evaluate the influence of NPK fertilization on production and leaf nutrient contents of eucalyptus cuttings in nutrient solution. The study was conducted from November 2008 to January 2009 in a greenhouse. The experimental design was completely randomized fractional factorial (4 x 4 x 4)½, with a total of 32 treatments with three replications. The treatments consisted of four doses of N (50, 100, 200 and 400 mg L-1) as urea, P (7.5, 15, 30 and 60 mg L-1) in the form of phosphoric acid and K (50, 100, 200 and 400 mg L-1) in the form of potassium chloride in the nutrient solution. Only the effect of N alone was significant for the number and dry weight of minicuttings per ministump, with a linear decreasing effect with increasing N levels. The highest number of cuttings was obtained at a dose of 50, 7.5 and 50 mg L-1 of N, P and K, respectively.