The Antiviral and Virucidal Activities of Voacangine and Structural Analogs Extracted from Tabernaemontana cymosa Depend on the Dengue Virus Strain.

Currently, no specific licensed antiviral exists for treating the illness caused by dengue virus (DENV). Therefore, the search for compounds of natural origin with antiviral activity is an important area of research. In the present study, three compounds were isolated and identified from seeds of Tabernaemontana cymosa plants. The in vitro antiviral effect of those compounds and voacangine against different DENV strains was assessed using different experimental approaches: compounds added before the infection (Pre), at the same time with the virus (Trans), after the infection (Post) or compounds present in all moments of the experiment (Pre-Trans-Post, Combined treatment). In silico studies (docking and molecular dynamics) were also performed to explain the possible antiviral mechanisms. The identified compounds were three structural analogs of voacangine (voacangine-7-hydroxyindolenine, rupicoline and 3-oxo-voacangine). In the Pre-treatment, only voacangine-7-hydroxyindolenine and rupicoline inhibited the infection caused by the DENV-2/NG strain (16.4% and 29.6% infection, respectively). In the Trans-treatment approach, voacangine, voacangine-7-hydroxyindolenine and rupicoline inhibited the infection in both DENV-2/NG (11.2%, 80.4% and 75.7% infection, respectively) and DENV-2/16681 infection models (73.7%, 74.0% and 75.3% infection, respectively). The latter strain was also inhibited by 3-oxo-voacangine (82.8% infection). Moreover, voacangine (most effective virucidal agent) was also effective against one strain of DENV-1 (DENV-1/WestPac/74) and against the third strain of DENV-2 (DENV-2/S16803) (48.5% and 32.4% infection, respectively). Conversely, no inhibition was observed in the post-treatment approach. The last approach (combined) showed that voacangine, voacangine-7-hydroxyindolenine and rupicoline inhibited over 90% of infections (3.5%, 6.9% and 3.5% infection, respectively) of both strains (DENV-2/NG and DENV-2/16681). The free energy of binding obtained with an in silico approach was favorable for the E protein and compounds, which ranged between -5.1 and -6.3 kcal/mol. Finally, the complex formed between DENV-2 E protein and the best virucidal compound was stable for 50 ns. Our results show that the antiviral effect of indole alkaloids derived from T. cymose depends on the serotype and the virus strain.

In Vitro and In Silico Anti-Arboviral Activities of Dihalogenated Phenolic Derivates of L-Tyrosine.

Despite the serious public health problem represented by the diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses, there are still no specific licensed antivirals available for their treatment. Here, we examined the potential anti-arbovirus activity of ten di-halogenated compounds derived from L-tyrosine with modifications in amine and carboxyl groups. The activity of compounds on VERO cell line infection and the possible mechanism of action of the most promising compounds were evaluated. Finally, molecular docking between the compounds and viral and cellular proteins was evaluated in silico with Autodock Vina®, and the molecular dynamic with Gromacs®. Only two compounds (TDC-2M-ME and TDB-2M-ME) inhibited both ZIKV and CHIKV. Within the possible mechanism, in CHIKV, the two compounds decreased the number of genome copies and in the pre-treatment strategy the infectious viral particles. In the ZIKV model, only TDB-2M-ME inhibited the viral protein and demonstrate a virucidal effect. Moreover, in the U937 cell line infected with CHIKV, both compounds inhibited the viral protein and TDB-2M-ME inhibited the viral genome too. Finally, the in silico results showed a favorable binding energy between the compounds and the helicases of both viral models, the NSP3 of CHIKV and cellular proteins DDC and ß2 adrenoreceptor.

In vitro and in silico anti-dengue activity of compounds obtained from Psidium guajava through bioprospecting.

BACKGROUND: For decades, bioprospecting has proven to be useful for the identification of compounds with pharmacological potential. Considering the great diversity of Colombian plants and the serious worldwide public health problem of dengue-a disease caused by the dengue virus (DENV)-in the present study, we evaluated the anti-DENV effects of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast, and 5 fractions and 5 compounds derived from Psidium guajava. METHODS: The cytotoxicity and antiviral effect of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast was evaluated in epithelial VERO cells. Five fractions were obtained by open column chromatography from the ethanolic extract with the highest selectivity index (SI) (derived from P. guajava, SI: 128.2). From the fraction with the highest selectivity (Pg-YP-I-22C, SI: 35.5), five compounds were identified by one- and two-dimensional nuclear magnetic resonance spectroscopy. The antiviral effect in vitro of the fractions and compounds was evaluated by different experimental strategies (Pre- and post-treatment) using non-toxic concentrations calculated by MTT method. The DENV inhibition was evaluated by plate focus assay. The results were analyzed by means of statistical analysis using Student's t-test. Finally the antiviral effect in Silico was evaluated by molecular docking. RESULTS: In vitro evaluation of these compounds showed that three of them (gallic acid, quercetin, and catechin) were promising antivirals as they inhibit the production of infectious viral particles via different experimental strategies, with the best antiviral being catechin (100% inhibition with a pre-treatment strategy and 91.8% with a post-treatment strategy). When testing the interactions of these compounds with the viral envelope protein in silico by docking, only naringin and hesperidin had better scores than the theoretical threshold of - 7.0 kcal/mol (- 8.0 kcal/mol and - 8.2 kcal/mol, respectively). All ligands tested except gallic acid showed higher affinity to the NS5 protein than the theoretical threshold. CONCLUSION: Even though bioprospecting has recently been replaced by more targeted tools for identifying compounds with pharmacological potential, our results show it is still useful for this purpose. Additionally, combining in vitro and in silico evaluations allowed us to identify promising antivirals as well as their possible mechanisms of action.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America.

Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America

ABSTRACT Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.

Actualización en diagnóstico del dengue: evolución de las técnicas y su aplicación real en la clínica / Update on dengue diagnosis: techniques evolution and their clinical application

Resumen: El dengue es la enfermedad viral transmitida por vectores de mayor importancia en el mundo. Debido a que hasta el momento no existe una vacuna licenciada para la prevención de la infección ni una terapia específica para controlar la enfermedad, el diagnóstico temprano y específico resulta ser una herramienta de vital importancia para brindar un tratamiento rápido y oportuno al paciente. Aunque hace más de seis décadas se dispone de una variedad de técnicas de laboratorio para el diagnóstico de esta enfermedad, es frecuente encontrar que noexista un consenso en el personal o en las entidades colombianas encargadas del diagnóstico de laboratorio sobre las ventajas y las limitaciones de estas técnicas, lo que dificulta dichodiagnóstico. Adicionalmente, debido a que la mayoría de técnicas que se usan se basan en la respuesta inmune frente al virus, y se pueden presentar reacciones cruzadas con otros virusgenéticamente relacionados, la posibilidad de falsos positivos es alta. Principalmente, por estarazón, en los últimos años ha aumentado el desarrollo y la validación de técnicas moleculares,ya que son más sensibles, específicas y rápidas que las técnicas celulares e inmunológicas.Teniendo en cuenta estos antecedentes, se hace necesario comparar a la luz de la literatura disponible la utilidad real de las técnicas de laboratorio existentes, clasificándolas en celulares,inmunológicas y moleculares.

Abstract: Dengue is the vector-borne viral disease of major importance in the world. Because so far there is no licensed vaccine that can be used to prevent the infection and there is no specific antiviral treatment, the early and specific diagnosis is a powerful tool to provide prompt and timely management of the patient. Despite during more than six decades there has been available a wide variety of laboratory techniques that can be used for the diagnosis of dengue, there is often lack of a consensus among the people or entities responsible of the diagnosis about the advantages and disadvantages of these techniques; for this reason, the diagnosis of dengue is more difficult. Moreover, taking into account that most of the techniques used are based on the immune response against dengue virus, and a cross reactivity with other viruses genetically related could be possible; the possibility of false positives is high. Accordingly, in recent years the development and validation of molecular techniques has increased, as they are more sensitive, specific and rapid than the cellular and immunological techniques. Therefore, it is necessary to compare the usefulness of laboratory techniques to dengue diagnosis, such as cellular, immunological and molecular tests.

Competencia vectorial: consideraciones entomológicas y suinfluencia sobre la epidemiología del dengue / Vector competence: entomological considerations and its implications on the epidemiology of dengue

Las enfermedades transmitidas por vectores constituyen un complejo problema de salud en el ámbito mundial, especialmente en áreas tropicales y subtropicales, que por sus condiciones ambientales favorecen la supervivencia del vector. El dengue es la principal enfermedad transmitida por vectores en el mundo, cuyo agente etiológico es el Virus Dengue, el cual es transmitido por la picadura de mosquitos hematófagos de la especie Aedes aegypti. La interacción virus-vector es esencial para una transmisión efectiva y depende de factores tanto virales como vectoriales. Entre los factores relacionados con el vector, la competencia vectorial es considerada de gran importancia, pues se refiere a la capacidad intrínseca del vector para infectarse con el virus, permitir su replicación y posteriormente su transmisión a un huésped susceptible. A su vez, la habilidad para ser un buen vector depende principalmente de barreras naturales a la infección, barreras inmunológicas y presencia de receptores específicos para el virus. Todas estas características, sumadas a las características propias del virus, favorecerán o no la transmisión del mismo. Un estudio integral dela relación virus-vector permitirá comprender sus implicaciones en la epidemiología de la enfermedad. Estos temas han sido revisados en el presente documento, discutiendo su importancia dentro del contexto entomológico y epidemiológico.

Vector-borne diseases are a serious problem in public health, especially in tropical and subtropical areas where environmental conditions favor the survival and expansion of vectors into new habitats. Dengue fever is one of the most important vector-borne diseases transmitted by arthropods (arbovirosis) worldwide. The etiologic agent of the disease is the dengue virus, which is transmitted by the bite of bloodsucking Aedes aegypti mosquitoes. The virus-vector interaction is essential for the efficient transmission of the disease, and depends on both, viral factors and vector competence or intrinsic vector capacity to be infected with the virus. Vector competence allows virus replication and subsequent transmission to susceptible hosts. Factors that influence vector capacity include: natural barriers to infection, immunological defenses as well as the presence of receptors for the virus. All these factors inaddition to the viral characteristics will determine the degree of transmission. There is a need for a better understanding of the virus-vector relationship and its epidemiological implications. These issues are addressed in this article.