Description of respiratory syncytial virus genotypes circulating in Colombia

Introduction: Respiratory syncytial virus (RSV) is the most common cause of acute respiratory infections in children younger than two years but also produces infection in older children and even reinfection in people of any age, a characteristic related to the existence of different infecting subtypes and genotypes. Although Colombia has established the surveillance of classical respiratory viruses, there is no information about the RSV genotypes circulating in Colombian patients. Methodology: A subgroup of 227 previously RSV positive respiratory secretion samples were taken from a nationwide surveillance study, amplified and sequenced to define the circulation pattern of RSV subtypes and genotypes during 2000-2009 period in Colombia. Results. RSV exhibited seasonal behavior with an A subtype more prevalent. Both RSV subtypes had low nucleotide variability. During the study period, the GA2 and GA5 genotypes from RSV subtype A and the BA genotype from RSV subtype B were found. Conclusion. In this report, for the first time RSV genotypes circulating in Colombia were described, this information adds valuable information about virus epidemiology helping to understand the RSV epidemic and prepare our country for the introduction of new vaccines

A tangled threesome: understanding arbovirus infection in Aedes spp. and the effect of the mosquito microbiota

Arboviral infections transmitted by Aedes spp. mosquitoes are a major threat to human health, particularly in tropical regions but are expanding to temperate regions. The ability of Aedes aegypti and Aedes albopictus to transmit multiple arboviruses involves a complex relationship between mosquitoes and the virus, with recent discoveries shedding light on it. Furthermore, this relationship is not solely between mosquitoes and arboviruses, but also involves the mosquito microbiome. Here, we aimed to construct a comprehensive review of the latest information about the arbovirus infection process in A. aegypti and A. albopictus, the source of mosquito microbiota, and its interaction with the arbovirus infection process, in terms of its implications for vectorial competence. First, we summarized studies showing a new mechanism for arbovirus infection at the cellular level, recently described innate immunological pathways, and the mechanism of adaptive response in mosquitoes. Second, we addressed the general sources of the Aedes mosquito microbiota (bacteria, fungi, and viruses) during their life cycle, and the geographical reports of the most common microbiota in adults mosquitoes. How the microbiota interacts directly or indirectly with arbovirus transmission, thereby modifying vectorial competence. We highlight the complexity of this tripartite relationship, influenced by intrinsic and extrinsic conditions at different geographical scales, with many gaps to fill and promising directions for developing strategies to control arbovirus transmission and to gain a better understanding of vectorial competence. The interactions between mosquitoes, arboviruses and their associated microbiota are yet to be investigated in depth

Humanized mice in dengue research: a comparison with other mouse models

Abstract: Dengue virus (DENV) is an arbovirus of the Flaviviridae family and is an enveloped virion containing a positive sense single-stranded RNA genome. DENV causes dengue fever (DF) which is characterized by an undifferentiated syndrome accompanied by fever, fatigue, dizziness, muscle aches, and in severe cases, patients can deteriorate and develop life-threatening vascular leakage, bleeding, and multi-organ failure. DF is the most prevalent mosquito-borne disease affecting more than 390 million people per year with a mortality rate close to 1% in the general population but especially high among children. There is no specific treatment and there is only one licensed vaccine with restricted application. Clinical and experimental evidence advocate the role of the humoral and T-cell responses in protection against DF, as well as a role in the disease pathogenesis. A lot of pro-inflammatory factors induced during the infectious process are involved in increased severity in dengue disease. The advances in DF research have been hampered by the lack of an animal model that recreates all the characteristics of this disease. Experiments in nonhuman primates (NHP) had failed to reproduce all clinical signs of DF disease and during the past decade, humanized mouse models have demonstrated several benefits in the study of viral diseases affecting humans. In DENV studies, some of these models recapitulate specific signs of disease that are useful to test drugs or vaccine candidates. However, there is still a need for a more complete model mimicking the full spectrum of DENV. This review focuses on describing the advances in this area of research

Dengue virus infection of blood-brain barrier cells: consequences of severe disease

More than 500 million people worldwide are infected each year by any of the four-dengue virus (DENV) serotypes. The clinical spectrum caused during these infections is wide and some patients may develop neurological alterations during or after the infection, which could be explained by the cryptic neurotropic and neurovirulent features of flaviviruses like DENV. Using in vivo and in vitro models, researchers have demonstrated that DENV can affect the cells from the blood–brain barrier (BBB) in several ways, which could result in brain tissue damage, neuronal loss, glial activation, tissue inflammation and hemorrhages. The latter suggests that BBB may be compromised during infection; however, it is not clear whether the damage is due to the infection per se or to the local and/or systemic inflammatory response established or activated by the BBB cells. Similarly, the kinetics and cascade of events that trigger tissue damage, and the cells that initiate it, are unknown. This review presents evidence of the BBB cell infection with DENV and the response established toward it by these cells; it also describes the consequences of this response on the nervous tissue, compares these evidence with the one reported with neurotropic viruses of the Flaviviridae family, and shows the complexity and unpredictability of dengue and the neurological alterations induced by it. Clinical evidence and in vitro and in vivo models suggest that this virus uses the bloodstream to enter nerve tissue where it infects the different cells of the neurovascular unit. Each of the cell populations respond individually and collectively and control infection and inflammation, in other cases this response exacerbates the damage leaving irreversible sequelae or causing death. This information will allow us to understand more about the complex disease known as dengue, and its impact on a specialized and delicate tissue like is the nervous tissue

Viability determination data for odontoblast-like cells exposed to resin monomers

Data in this article are associated with our research article “Dental Resin Monomers Induce Early and Potent Oxidative Damage on Human Odontoblast-like Cells.” Dental adhesives are polymeric compounds consisting of several chemical substances, including resin monomers, such as 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA), together with other comonomers, making up the organic matrix of the adhesive and whose composition is based on the methyl methacrylate chemistry. The release of residual monomers, susceptible to biodegradation, acts as a source of bioactive compounds, which can interact with tissues and induce a cytotoxic cellular response. The most used techniques to evaluate cytotoxicity, proliferation, or metabolic activity of cells exposed to different substances, are MTT and resazurin. Each chemistry evaluates cell viability differently, so the data obtained could vary depending on the technique sensitivity to detect changes in cell metabolism. The objective of this article was to present viability data as a function of the metabolic activity in human odontoblast-like cells (hOLCs), exposed to 3, 6, 9, and 12 mM HEMA, or 0.75, 1.5, 3, and 6 mM TEGDMA evaluated by the MTT, and resazurin techniques in the first 24 hours of exposure, at different time points. The absorbance data for the MTT test and the fluorescence intensity for the resazurin test were obtained by spectrometry. SIMSTAT software 2.6.5 for Windows was used to confirm the normal data distribution (Levene's test). Subsequently, an analysis of variance (one-way ANOVA) was performed to compare the control with each HEMA and TEGDMA concentration. Where a p < 0.05 indicated a high F value, a Fisher's least significant differences post-hoc analysis was performed, using an alpha value < 0.05. Data from the different time points were compared with a Student's t-test for each concentration. These data may be useful to compare the cytotoxic response of hOLCs with other cell types or the cell response to other resin monomers

In vivo differential susceptibility of sensory neurons to rabies virus infection

There is controversy with regard to the entry pathway of the rabies virus (RABV) into the central nervous system (CNS). Some authors have suggested that the virus inoculated at the periphery is captured and transported to CNS only by motor neurons; however, it has been reported that dorsal root ganglia (DRG) sensory neurons capture and transport the virus to the spinal cord (SC) and then to the brain. It is probable that preferences for one pathway or another depend on the site of inoculation and the post-infection time. Therefore, in the present study, we evaluated different vertebral segments and post-infection times, along with the location, number, and subpopulation of sensory neurons susceptible to infection after inoculating RABV in the footpads of adult mice. It was noted that the virus inoculated in the footpad preferentially entered the CNS through the large-sized DRG sensory neurons, while infection of the motor neurons occurred later. Further, it was found that the virus was dispersed in spinal cord trans-synaptically through the interneurons, arriving at both sensory neurons and contralateral motor neurons. In conclusion, we observed that RABV inoculated in the plantar footpad is captured preferentially by large sensory neurons and is transported to the DRG, where it replicates and is spread to the SC using transynaptic jumps, infecting sensory and motor neurons at the same level before ascending to the brain

In vivo infection by a neuroinvasive neurovirulent dengue virus

Although neurological manifestations associated with dengue infections have been reported in endemic countries, the viral or host characteristics determining the infection or alteration of nervous function have not been described. In order to investigate neurobiological conditions related to central nervous system dengue virus (DENV) infection, we established a mouse model of neuroinfection. A DENV-4 isolate was first adapted to neuroblastoma cells, later inoculated in suckling mice brain, and finally, this D4MB-6 viral variant was inoculated intraperitoneally in Balb/c mice at different postnatal days (pnd). Virusinduced fatal encephalitis in 2 and 7 pnd mice but infected at 14 and 21 pnd mice survived. The younger mice presented encephalitis at the sixth day postinfection with limb paralysis and postural instability concomitant with efficient viral replication in brain. In this mice model, we found activated microglial cells positive to viral antigen. Neurons, oligodendrocytes, and endothelial cells were also infected by the D4MB-6 virus in neonatal mice, which showed generalized and local plasma leakage with blood-brain barrier (BBB) severe damage. These results suggest that there was a viral fitness change which led to neuroinfection only in immune or neurological immature mice. Infection of neurons, endothelial, and microglial cells may be related to detrimental function or architecture found in susceptible mice. This experimental neuroinfection model could help to have a better understanding of neurological manifestations occurring during severe cases of dengue infection

Description of high rates of unapparent and simultaneous multiple dengue virus infection in a Colombian jungle settlement

Dengue disease statistics is mainly based on consulting patients with febrile illness, but misdiagnosed and asymptomatic cases are important to measure dengue epidemiology in endemic areas. The main objective of this work was to determine the prevalence of IgM and IgG antibodies or NS1 antigen and viral RNA in a group of healthy volunteers from an isolated village in Colombian Chocó rain forest. It found 51.7% of virologically PCR confirmed asymptomatic cases, despite low IgM seroprevalence. It was confirmed that all four serotypes are in the circulation and in 17.2% of individuals it detected natural coinfections of two or three different serotypes simultaneously. This is the first report in Colombia evaluating viremia in asymptomatic volunteers. These findings pose a big concern about the transmission of dengue virus by asymptomatic individuals because they can spread the virus without taking appropriate control measures

Assembly and release of dengue virus: Role of alix protein

Algunos virus envueltos usurpan la maquinaria celular ESCRT (complejo de clasificación endosomal requerido para el transporte) para llevar a cabo funciones como la transcripción, la traducción, el ensamblaje y la liberación de partículas virales desde las células huésped. Aunque esta estrategia ha sido estudiada principalmente en retrovirus, son varios los virus envueltos que la usan. El objetivo del trabajo fue explorar la participación de una proteína accesoria de ESCRT, la proteína Alix, en la transcripción, traducción, ensamblaje y liberación del virus dengue (DENV), así como su interacción con la proteína viral NS3. Células A549 infectadas con DENV2 fueron tratadas con pequeños ARN de interferencia (siRNA) para disminuir la expresión (“knock-down”) de la proteína Alix. Simultáneamente, se obtuvo una línea A549 que expresaba una proteína NS3 recombinante y sobre este sistema se hicieron ensayos de inmunoprecipitación y “pull-down” para detectar interacción entre NS3 y Alix. Los resultados mostraron que el “knock-down” de Alix no tuvo efecto notable en la transcripción o la traducción viral, pero sí en el ensamblaje y la liberación de DENV2, mientras que los ensayos de “pull-down” revelaron la interacción entre NS3 y Alix. La participación de Alix en la producción de DENV2 y su interacción con NS3 constituyen un potencial blanco para el diseño de estrategias dirigidas a controlar la propagación de DENV.Since the finding that HIV recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to accomplish viral budding, this strategy has emerged as an escape route for enveloped viruses also. The work aimed to explore the participation of the cellular protein Alix (a human protein that acts as an adapter in the ESCRT pathway) on the transcription, protein expression, assembly and release of Dengue virus (DENV), and explore for its potential interaction with the viral protein NS3. To this purpose, A549 cells were infected with DENV2 and treated with small interfering RNAs (siRNA) to generate an Alix stable knockdown cells line. Also, an A549 cells line expressing a histidine-tagged NS3 protein was obtained. Both cells lines were used in immunoprecipitation and pull-down assays to assess the interaction between NS3 and Alix. The results showed that Alix knockdown had no effect on viral transcription or viral protein expression but influenced the assembly and release of DENV2 negatively. Finally, pull-down assays revealed the interaction between NS3 and Alix. The finding of an Alix participation in the production of DENV2 and its interaction with NS3 provides a potential target for the design of control/inhibition strategies against DENV spread

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses. • Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies. • Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species. • A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission