Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform.

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription.

Structure of a protein-detergent complex: the balance between detergent cohesion and binding.

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein-detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.

Genetic make up and structure of Colombian populations by means of uniparental and biparental DNA markers.

Colombia is a country with great geographic heterogeneity and marked regional differences in pre-Columbian native population density and in the extent of past African and European immigration. As a result, Colombia has one of the most diverse populations in Latin America. Here we evaluated ancestry in over 1,700 individuals from 24 Colombian populations using biparental (autosomal and X-Chromosome), maternal (mtDNA), and paternal (Y-chromosome) markers. Autosomal ancestry varies markedly both within and between regions, confirming the great genetic diversity of the Colombian population. The X-chromosome, mtDNA, and Y-chromosome data indicate that there is a pattern across regions indicative of admixture involving predominantly Native American women and European and African men.

Ptuba: a tool for the visualization of helix surfaces in proteins.

Projection of transmembrane helices using a Uniform B-spline Algorithm is a tool for the visualization of interactions between helices in membrane proteins. It allows the user to generate projections of 3D helices, no matter what their deviations from a canonical helix might be. When associated with adapted coloring schemes it facilitates the comprehension of helix-helix interactions. Examples of transmembrane proteins were chosen to illustrate the advantages that this method provides. In the glycophorin A dimer we can easily appreciate the structural features behind homodimerisation. Using the structure of the fumarate reductase we analyze the contact surfaces inside a helical bundle and thanks to structures from a molecular dynamics simulation we see how modifications in structure and electrostatics relate to their interaction. We propose the use of this tool as an aid to the visualization and analysis of transmembrane helix surfaces and properties.

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome.

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

Occupational history of exposure to zoonotic agents in people dedicated to livestock in San Pedro de los Milagros, Antioquia, Colombia / Antecedentes ocupacionales de la exposición a agentes zoonóticos en personas dedicadas a la ganadería en San Pedro de los Milagros, Antioquia, Colombia

Abstract Introduction: According to the World Health Organization, livestock farming is one of the anthropic activities in which workers are exposed to various zoonotic agents. Objectives: To establish the frequency of seropositivity (IgG antibodies) against some zoonotic agents in people with occupational exposure to livestock in San Pedro de los Milagros (Antioquia), and to analyze associated factors. Materials and methods: Descriptive study carried out on a population of 328 cattle farmers. Demographic data were collected and the seropositivity frequency of IgG antibodies to Babesia bovis, Babesia bigemina, Anaplasma phagocytophilum, Ehrlichia chaffensis, Borrelia burgdorferi, Coxiella burnetii, Francisella tularensis, Brucella abortus, Brucella suis, Leptospira interrogans, and Toxoplasma gondii was determined. Overall and specific prevalence, prevalence ratios and binary logistic regressions were estimated. Results: The highest seropositivity frequencies were 47.6% for T. gondii, 33.5% for B. burgdorferi and 13% for E. chaffensis. The prevalence of T. gondii and B. burgdorferi had statistical association with sex [RP:1.3 (CI:1.0-1.8) and 2.0 (CI:1.1-3.9) respectively], and age group [(RP:1.5 (CI:1.2-1,9) and 2.5 (CI:1.4-6.4) respectively]. In workers with more than 10 years of related work experience, statistical association was 50% [RP:1.5 (CI:1.2-1.9) and 2.5 (CI:1.6-2.3), respectively]. There were no seropositive results for B. abortus, B. suis, B. bovis and B. bigemina. Conclusions: Exposure to some zoonotic agents was evidenced. This is determinant for the knowledge of tropical zoonotic diseases transmitted by vectors in livestock production systems.

Resumen Introducción. Según la Organización Mundial de la Salud, la ganadería es una actividad antrópica profesional en la que los trabajadores se ven expuestos a diversos agentes zoonóticos. Objetivos. Determinar la frecuencia de seropositividad (anticuerpos IgG) frente a algunos agentes zoonóticos en personas con exposición ocupacional a la ganadería en San Pedro de los Milagros (Antioquia) y analizar los factores asociados. Materiales y métodos. Estudio descriptivo realizado en una población de 328 productores ganaderos. Se recolectaron datos demográficos; se determinó la frecuencia de seropositividad de anticuerpos IgG de Babesia bovis, Babesia bigemina, Anaplasma phagocytophilum, Ehrlichia chaffensis, Borrelia burgdorferi, Coxiella burnetii, Francisella tularensis, Brucella abortus, Brucella suis, Leptospira interrogans y Toxoplasma gondii, y se calcularon prevalencias globales y específicas, razones de prevalencia y regresiones logísticas binarias. Resultados. Las frecuencias más altas de seropositividad fueron 47.6% para T. gondii, 33.5% para B. burgdorferi y 13% para E. chaffensis. Las prevalencias de T. gondii y B. burgdorferi presentaron asociación estadística con el sexo (RP: 1.3 (IC: 1.0-1.8) y 2.0 (IC: 1.1-3.9), respectivamente) y el grupo etario (RP:1.5 (IC: 1.2-1.9) y 2.5 (IC: 1.4-6.4) respectivamente). En trabajadores con más de diez años en la labor la asociación estadística fue de 50% (RP:1.5 (IC:1.2-1.9) y 2.5 (IC:1.6-2.3), respectivamente). No hubo resultados de seropositividad para B. abortus, B. suis, B. bovis y B. bigemina. Conclusiones. Se evidenció exposición a algunos agentes zoonóticos, lo que resulta determinante para el conocimiento de las enfermedades zoonóticas tropicales transmitidas por vectores en la ganadería.