New insights into the antiviral activity of nordihydroguaiaretic acid: Inhibition of dengue virus serotype 1 replication.

BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.

Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner.

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

Algal and Cyanobacterial Lectins and Their Antimicrobial Properties.

Lectins are proteins with a remarkably high affinity and specificity for carbohydrates. Many organisms naturally produce them, including animals, plants, fungi, protists, bacteria, archaea, and viruses. The present report focuses on lectins produced by marine or freshwater organisms, in particular algae and cyanobacteria. We explore their structure, function, classification, and antimicrobial properties. Furthermore, we look at the expression of lectins in heterologous systems and the current research on the preclinical and clinical evaluation of these fascinating molecules. The further development of these molecules might positively impact human health, particularly the prevention or treatment of diseases caused by pathogens such as human immunodeficiency virus, influenza, and severe acute respiratory coronaviruses, among others.

Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder.

Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

Human Metapneumovirus: Epidemiology and genotype diversity in children and adult patients with respiratory infection in Córdoba, Argentina.

Human Metapneumovirus (hMPV) is responsible for acute respiratory infections in humans, with clinical and epidemiological relevance in pediatric, elderly, and immunocompromised populations. These features are largely unknown in Córdoba, Argentina and in adults in general. Hence, our goal was to broadly characterize hMPV infection in patients of all ages hospitalized with acute respiratory infections in Córdoba, Argentina, including epidemiology, clinical features and genetic diversity. Nasopharyngeal secretions were obtained from 795 patients during 2011-2013, 621 patients were 0-25 years old and 174 were 26-85 years old. HMPV was assayed by RT-PCR and other respiratory viruses by indirect immunofluorescence. Local strains were identified by sequence analysis. Human Metapneumovirus was detected in 20.3% (161/795) patients, 13.1% as single infections and 7.2% in co-infections, more frequently with Respiratory Syncytial Virus. HMPV circulated during late winter and spring in all age patients, but mainly in children under 4 years old in 71.4% (115/161) and adults between 26 and 59 years old in 12.4% (20/161). The most prevalent diagnosis was mild acute respiratory infection in 59.6% (96/161) and bronchiolitis in 9.3% (15/161). Local strains were clustered within A2 subtype; they presented 73-100% identities among them, showing a high degree of homology compared to isolations from neighboring countries. We demonstrate that hMPV circulated among all age patients with respiratory infection during 2011-2013 in Córdoba, contributing to the understanding of this virus, its diagnosis and patient handling in local health-care centers.

Long-term impact of chronic variable stress in adolescence versus adulthood.

Adolescence is a period of active development of stress regulatory neurocircuitry. As a consequence, mechanisms that control the responses to stress are not fully matured during this developmental period, which may result in vulnerability to chronic stress. We hypothesized that adolescent chronic stress would have negative consequences on stress adaptation later in life. Male Wistar rats (PND40) were subjected to chronic variable stress (CVS) for 2 weeks, with 2 daily stressors randomly presented and overnight social stressors twice a week. After five weeks, animals were evaluated during adulthood, using the elevated plus maze (EPM) and the forced swim test (FST). The hypothalamic-pituitary adrenal (HPA) axis response to a 30-min restraint was also assessed. Results are compared to those of adult rats tested 5 weeks following CVS cessation. Our results demonstrate that the long-term effects of CVS are specific to the age of application of the stress regime. We show how behavior and HPA axis response as well as hypothalamic paraventricular nucleus activation can differ with age, resulting in differential behavioral adaptations for animals stressed in adolescence and dysregulation of the HPA axis in the animals stressed in adulthood, These data underscore the importance of the adolescent period in determining resilience of the HPA axis and programming behavioral responses later in life.

Pixuna virus modifies host cell cytoskeleton to secure infection.

Pixuna virus (PIXV) is an enzootic member of the Venezuelan Equine Encephalitis Virus complex and belongs to the New World cluster of alphaviruses. Herein we explore the role of the cellular cytoskeleton during PIXV replication. We first identified that PIXV undergoes an eclipse phase consisting of 4 h followed by 20 h of an exponential phase in Vero cells. The infected cells showed morphological changes due to structural modifications in actin microfilaments (MFs) and microtubules (MTs). Cytoskeleton-binding agents, that alter the architecture and dynamics of MFs and MTs, were used to study the role of cytoskeleton on PIXV replication. The virus production was significantly affected (p < 0.05) after treatment with paclitaxel or nocodazole due to changes in the MTs network. Interestingly, disassembly of MFs with cytochalasin D, at early stage of PIXV replication cycle, significantly increased the virus yields in the extracellular medium (p < 0.005). Furthermore, the stabilization of actin network with jasplakinolide had no effect on virus yields. Our results demonstrate that PIXV relies not only on intact MTs for the efficient production of virus, but also on a dynamic actin network during the early steps of viral replication.

[Monoinfection of human Metapneumovirus in Cordoba: first clinical and epidemiological research in children with respiratory infection in 2011]. / Monoinfección de Metapneumovirus humano en Córdoba: primeras investigaciones clínico-epidemiológicas en niños con infección respiratoria en 2011.

Human metapneumovirus (hMPV) RNA virus discovered in 2001, is a pathogen associated with acute respiratory infection (ARI) in children under 5 years; its prevalence ranges from 5-15%. In Córdoba, it is not integrated into the viral research in patients with low IRA (LARI). OBJECTIVE: Detect hMPV in children under 5 years hospitalized for LARI in the Children's Hospital "Santísima Trinidad" of Cordoba (HNC) in 2011 and describe the clinical and epidemiological characteristics of monoinfecciones without comorbidity. POPULATION AND METHOD: Prospective, observational study. It includes (informed consent) children under 5 years with LARI of HNC from January to December 2011. The viral detection was performed using immunofluorescence of nasopharyngeal aspirate secretions. Demographic, epidemiological and clinical data of positive cases were recorded. RESULTS: Of 223 patients enrolled, respiratory viruses were detected in 74 (33.2%). HMPV prevalence was 4.04% (9/223), representing the 2nd place with Parainfluenza 3 (4.04%) after RSV (19.73%). Season from July to December. The average age for hMPV was 7.4 ± 6.8 months (0-60 months), 4/9 males. The average hospital stay in days was 5.6 ± 0.5 and prodrome days: 1.9 days ± 0.6. All patients require oxygen therapy (3.9 ± 1.3 days) without mechanical ventilation. Diagnosis of bronchiolitis cases occurred in 5/9 and 4/9 pneumonia. No complications at discharge. CONCLUSIONS: First report to document the presence of hMPV in child population of Cordoba. Its prevalence in 2011 was 4, 04%. Among monoinfecciones no fatalities or complications at discharge were recorded.

Metapneumovirus humano (MPVh), virus ARN descubierto en 2001, es un patógeno relacionado con infección respiratoria aguda (IRA) en menores de 5 años; su prevalencia oscila entre el 5-15%. En Córdoba no está integrado a la pesquisa viral en pacientes con IRA baja (IRAB). Objetivo. Detectar MPVh en menores de 5 años hospitalizados por IRAB en el Hospital de Niños de la Santísima Trinidad de Córdoba (HNC) durante el 2011 y describir características clínico-epidemiológicas de las monoinfecciones sin comorbilidad previa. Población y método. Estudio prospectivo, observacional. Participaron (consentimiento informado) menores de 5 años con IRAB del HNC desde enero a diciembre de 2011. La detección viral se realizó con Inmunofluorescencia de aspirado de secreciones nasofaríngeas. Se registraron datos demográficos, epidemiológicos y clínicos de los casos positivos. Resultados. De 223 pacientes incluidos, se detectó algún virus respiratorio en 74 (33,2%). La prevalencia de MPVh fue de 4,04% (9/223), representando el 2° lugar con Parainfluenza 3 (4,04 %), luego de VRS (19,73%). Estacionalidad julio-diciembre. La edad media para MPVh fue de 7,4±6,8 meses (0 a 60 meses), 4/9 varones. La media de hospitalización fue de 5,6±0,5 días, y de pródromo 1,9±0,6 días. Todos requirieron oxigenoterapia (3,9±1,3 días) sin asistencia respiratoria mecánica. Diagnóstico de bronquiolitis en 5/9 casos y neumonía en 4/9. Sin complicaciones al alta. Conclusiones. Primer trabajo en documentar la presencia de MPVh en población infantil de Córdoba. Su prevalencia durante el 2011 fue del 4, 04 %. Entre las monoinfecciones no se registraron casos fatales ni complicaciones al momento del alta.

Odor-avoidance or odor-preference induced by amphetamine in the infant rat depending on the dose and testing modality.

By the second postnatal week of life infant rats can acquire taste avoidance induced by amphetamine. Psychostimulant drugs supports appetitive and aversive learning in adult rats. Their appetitive effects are more likely to become associated with contextual cues, while the aversive ones have been consistently found in taste aversion learning. To explain this paradox, it has been proposed that rats would avoid a taste that predicts a change in their homeostasis because this species cannot vomit. In this study we assessed the motivational properties of amphetamine in preweanling rats by means of an odor conditioning preparation, which enables the analysis of the hedonic value of the memory by means of a consumption test or in terms of locomotor approach to the odor. Results indicate that regardless of the amphetamine dose (1 or 5 mg/kg), when animals were evaluated in the intake test, subjects avoided the odor. However, the outcome in the locomotor avoidance test varied as a function of the amphetamine dose. Rats trained with the low dose (1 mg/kg) showed odor preference, while the highest amphetamine dose (5 mg/kg) induced odor avoidance. When LiCl was employed as an unconditioned stimulus (US), rats showed avoidance in the intake and locomotor activity tests. These data indicate that amphetamine, like other drugs of abuse, supports appetitive conditioning in preweanling rats. Interestingly, infant rats expressed conditioned odor avoidance or preference depending on the dose and testing modality. Results were discussed considering current theories of avoidance learning induced by rewarding drugs.

Dopamine receptors modulate ethanol's locomotor-activating effects in preweanling rats.

Near the end of the second postnatal week motor activity is increased soon after ethanol administration (2.5 g/kg) while sedation-like effects prevail when blood ethanol levels reach peak values. This time course coincides with biphasic reinforcement (appetitive and aversive) effects of ethanol determined at the same age. The present experiments tested the hypothesis that ethanol-induced activity during early development in the rat depends on the dopamine system, which is functional in modulating motor activity early in ontogeny. Experiments 1a and 1b tested ethanol-induced activity (0 or 2.5 g/kg) after a D1-like (SCH23390; 0, .015, .030, or .060 mg/kg) or a D2-like (sulpiride; 0, 5, 10, or 20 mg/kg) receptor antagonist, respectively. Ethanol-induced stimulation was suppressed by SCH23390 or sulpiride. The dopaminergic antagonists had no effect on blood ethanol concentration (Experiments 2a and 2b). In Experiment 3, 2.5 g/kg ethanol increased dopamine concentration in striatal tissue as well as locomotor activity in infant Wistar rats. Adding to our previous results showing a reduction in ethanol induced activity by a GABA B agonist or a nonspecific opioid antagonist, the present experiments implicate both D1-like and D2-like dopamine receptors in ethanol-induced locomotor stimulation during early development. According to these results, the same mechanisms that modulate ethanol-mediated locomotor stimulation in adult rodents seem to regulate this particular ethanol effect in the infant rat.