Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.

Antiviral activity of myricetin glycosylated compounds isolated from Marcetia taxifolia against chikungunya virus.

The chikungunya virus (CHIKV) has produced epidemic outbreaks of significant public health impact. The clinical symptoms of this disease are fever, polyarthralgia, and skin rash, generally self-limiting, although patients may develop a chronic disabling condition or suffer lethal complications. Unfortunately, there is no specific treatment or vaccine available. Thus, the search for effective therapies to control CHIKV infection is an urgent need. This study evaluated the antiviral activity of flavonoids isolated from Marcetia taxifolia by in vitro and in silico analysis. Cytotoxicity of compounds was determined by MTT assay and viral load was assessed in cell substrates supernatants by plaque-forming and RT-qPCR assays. Selected molecules were analyzed by molecular docking assays. Myricetin 3-rhamnoside (MR) and myricetin 3-(6-rhamnosylgalactoside) (MRG) were tested for antiviral assays and analyzed by the TCID50 method and RT-qPCR. MR exhibited dose-dependent antiviral activity, reducing viral titer at concentrations of 150-18.8 µg/mL by at least 1-log. Similarly, MRG showed a significant decrease in viral titer at concentrations of 37.5, 9.4, and 2.3 µg/mL. RT-qPCR analysis also displayed a substantial reduction of CHIKV RNA for both flavonoids. Furthermore, molecular docking of the selected flavonoids proposed the nsP3 macrodomain as a possible target of action. Our study reveals that MR and MRG could be considered promising anti-CHIKV therapeutic agents. Molecular modeling studies showed MR and MRG ligands with a high affinity for the N-terminal region of the nsP3 macrodomain, postulating them as a potential target of action for the CHIKV control.

Comparison of Four Real-Time Polymerase Chain Reaction Assays for the Detection of SARS-CoV-2 in Respiratory Samples from Tunja, Boyacá, Colombia.

Coronavirus disease (COVID-19) is an infectious disease caused by SARS-CoV-2. In Colombia, many commercial methods are now available to perform the RT-qPCR assays, and laboratories must evaluate their diagnostic accuracy to ensure reliable results for patients suspected of being positive for COVID-19. The purpose of this study was to compare four commercial RT-qPCR assays with respect to their ability to detect the SARS-CoV2 virus from nasopharyngeal swab samples referred to Laboratorio Carvajal IPS, SAS in Tunja, Boyacá, Colombia. We utilized 152 respiratory tract samples (Nasopharyngeal Swabs) from patients suspected of having SARS-CoV-2. The diagnostic accuracy of GeneFinderTM COVID-19 Plus RealAmp (In Vitro Diagnostics) (GF-TM), One-Step Real-Time RT-PCR (Vitro Master Diagnostica) (O-S RT-qPCR), and the Berlin modified protocol (BM) were assessed using the gold-standard Berlin protocol (Berlin Charité Probe One-Step RT-qPCR Kit, New England Biolabs) (BR) as a reference. Operational characteristics were estimated in terms of sensitivity, specificity, agreement, and predictive values. Using the gold-standard BR as a reference, the sensitivity/specificity of the diagnostic tests was found to be 100%/92.7% for GF-TM, 92.75%/67.47% for O-S RT-qPCR, and 100%/96.39% for the BM protocol. Using BR as a reference, the sensitivity/specificity for the diagnostic tests were found to be 100%/92.7% for the GF-TM assay, 92.72%/67.47% for the O-S RT-qPCR, and 100%/96.39% for BM. Relative to the BR reference protocol, the GF-TM and BM RT-PCR assays obtained similar results (k = 0.92 and k = 0.96, respectively), whereas the results obtained by O-S-RT-qPCR were only moderately similar. We conclude that the GF-TM and BM protocols offer the best sensitivity and specificity, with similar results in comparison to the gold-standard BR protocol. We recommend evaluating the diagnostic accuracy of the OS-RT-qPCR protocol in future studies with a larger number of samples.

Inverse Molecular Docking Study of NS3-Helicase and NS5-RNA Polymerase of Zika Virus as Possible Therapeutic Targets of Ligands Derived from Marcetia taxifolia and Its Implications to Dengue Virus.

Dengue and Zika are two mosquito-borne diseases of great impact on public health around the world in tropical and subtropical countries. DENV and ZIKV belong to the Flaviviridae family and the Flavivirus genus. Currently, there are no effective therapeutic agents to treat or prevent these pathologies. The main objective of this work was to evaluate potential inhibitors from active compounds obtained from Marcetia taxifolia by performing inverse molecular docking on ZIKV-NS3-helicase and ZIKV-NS5-RNA polymerase as targets. This computational strategy is based on renormalizing the binding scores of the compounds to these two proteins, allowing a direct comparison of the results across the proteins. The crystallographic structures of the ZIKV-NS3-helicase and ZIKV-NS5-RNA-polymerase proteins share a great similarity with DENV homologous proteins. The P-loop active site of the crystallographic structure of ZIKV-NS3-helicase presents a high percentage of homology with the four dengue serotypes. It was found that most ligands of the active compounds (5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (5DP); 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HH); myricetin-3-O-rhamnoside (M3OR)) from Marcetia taxifolia had a better affinity for ZIKV-NS3-helicase than for ZIKV-NS5-RNA polymerase, as indicated by the negative multiple active site correction (MASC) score, except for M3RG that showed a higher affinity for ZIKV-NS5-RNA polymerase. On the other hand, the AutoDock Vina scores showed that M3OR had the highest score value (-9.60 kcal/mol) and the highest normalized score (1.13) against ZIKV-NS3-helicase. These results in silico demonstrated that the nonstructural proteins NS3-helicase and NS5-RNA polymerase, which share similar molecular structures between the selected viruses, could become therapeutic targets for some bioactive compounds derived from Marcetia taxifolia.

Molecular characteristics and replication mechanism of dengue, zika and chikungunya arboviruses, and their treatments with natural extracts from plants: An updated review.

Viruses transmitted by arthropods (arboviruses) are the etiological agents of several human diseases with worldwide distribution; including dengue (DENV), zika (ZIKV), yellow fever (YFV), and chikungunya (CHIKV) viruses. These viruses are especially important in tropical and subtropical regions; where, ZIKV and CHIKV are involved in epidemics worldwide, while the DENV remains as the biggest problem in public health. Factors, such as, environmental conditions promote the distribution of vectors, deficiencies in health services, and lack of effective vaccines, guarantee the presence of these vector-borne diseases. Treatment against these viral diseases is only palliative since available therapies formulated lack to demonstrate specific antiviral activity and vaccine candidates fail to demonstrate enough effectiveness. The use of natural products, as therapeutic tools, is an ancestral practice in different cultures. According to WHO 80 % of the population of some countries from Africa and Asia depend on the use of traditional medicines to deal with some diseases. Molecular characteristics of these viruses are important in determining its cellular pathogenesis, emergence, and dispersion mechanisms, as well as for the development of new antivirals and vaccines to control strategies. In this review, we summarize the current knowledge of the molecular structure and replication mechanisms of selected arboviruses, as well as their mechanism of entry into host cells, and a brief overview about the potential targets accessed to inhibit these viruses in vitro and a summary about their treatment with natural extracts from plants.

Caracterización citogenética de lucilia sericata (Meigen, 1826) (Diptera: calliphoridae), Cepa Bogotá, Colombia / Cytogenetic characterization of lucilia sericata (Meigen, 1826) (diptera: calliphoridae), Bogotá-Colombia strain / Caracterização citogenética de lucilia sericata (Meigen, 1826) (diptera: calliphoridae), cepa Bogotá, Colômbia

Introducción: Lucilia sericata es una especie de importancia médica y forense, utilizada en terapia larval para curar heridas crónicas y en estudios médico-legales empleada en la estimación del intervalo post mórtem y el traslado de cadáveres. No existen registros de las características citogenéticas de esta mosca en el neotrópico. El objetivo principal de este trabajo fue identificar las características morfométricas cromosómicas y las estructuras primarias del cariotipo, a partir de especímenes de L. sericata de la cepa Bogotá, Colombia. Materiales y métodos: Se tomaron huevos embrionados, que fueron previamente esterilizados en su superficie, se maceraron y luego fueron sembrados en el medio de cultivo L-15, suplementado con 20% de SFB, e incubados a una temperatura de 28 °C, sin atmosfera de C0(2). La preparación de los cromosomas se obtuvo de monocapas celulares semiconfluentes, empleando diversas soluciones: antimitótica (Colchicina), hipotónica (KCl 0,075 M) y fijadora (Carnoy: metanol y ácido acético; 3:1). Se llevó a cabo la técnica de bandeo C para la identificación de regiones cromosómicas de heterocromatina constitutiva. Resultados: Se obtuvieron parámetros morfométricos de cada par cromosómico. El número diploide del cariotipo obtenido de los cultivos celulares fue 2n = 12; éstos se clasificaron morfológicamente, de acuerdo con patrones previamente establecidos, así: los pares I, II, IV y V fueron metacéntricos, y el par III fue submetacéntrico. A su vez, el par sexual fue heteromórfico, siendo el cromosoma X metacéntrico y el cromosoma Y submetacéntrico. El bandeo C fue positivo para todos los pares cromosómicos. Conclusiones: Se establecieron las características citogenéticas de L. sericata, cepa Bogotá, Colombia, relacionadas con número, forma, tamaño, posición del centrómero y regiones heterocromáticas de los cromosomas.

Objective: Lucilia sericata is an important species for medical and forensic purposes, it is used in maggot therapy in the treatment of chronic wounds and in medical-legal studies for establishing the post-mortem interval and the transfer of corpses. Currently there are no records of the cytogenetic characteristics of this fly in Neotropical region. The main objective of this study was to identify morphometric characteristics and primary structures from karyotype of L. sericata strain Bogota, Colombia. Methods and materials: Embryonated eggs were taken, which were previously surface sterilized, macerated and then seeded in L-15 medium culture, supplemented with 20% FBS and incubated at 28 °C, without C0(2) atmosphere. The preparation of chromosomes was obtained from semiconfluent monolayers, pretreated with various solutions: antimitotic (Colchicine), hypotonic (KCl 0.075 M) and fixative (Carnoy, methanol and acetic acid, 3:1). C-banding technique was carried out to identify chromosomal regions of constitutive heterochromatin. Results: Morphometric parameters were obtained from each pair of chromosomes. The diploid karyotype number obtained from cell cultures was 2n = 12; they were classified morphologically, according to patterns established previously, as follows: pairs I, II, IV and V were metacentric and pair III was submetacentric. On the other hand, the sexual pair was heteromorphic, being X chromosome metacentric and Y chromosome submetacentric. C banding was positive for all chromosome pairs. Conclusions: The cytogenetic characteristics of L. sericata, strain Bogotá, were established according to number, shape, centromer position and heterochromatic regions.

Introdução: Lucilia sericata é uma espécie de importência médica e forense, utilizada em terapia larval para curar feridas crônicas e em estudos médico-legais empregada na estimação do intervalo post morteme o traslado de cadáveres. Não existem registros das características citogéneticas desta mosca no neotrópico. O objetivo principal deste trabalho foi identificar as características morfométricas cromossômicas e as estruturas primárias do cariótipo, a partir de especímenes de L. sericata da cepa Bogotá, Colômbia. Materiais e métodos: Tomaram-se ovos embrionados, que foram previamente esterilizados em sua superfície, se maceraram e depois foram semeados no médio de cultivo L-15, suplementado com 20% de SFB, e incubados a uma temperatura de 28 °C, sem atmosfera de C02. A preparação dos cromossomas obteve-se de monocamadas celulares semiconfluentes, utilizando diversas soluções: antimitótica (Colchicina), hipotônica (KCl 0,075 M) e fixadora (Carnoy: metanol y ácido acético; 3:1). Levou-se a cabo a técnica de bandas C para a identificação de regiões cromossômicas de heterocromatina constitutiva. Resultados: Se obtiveram parâmetros morfométricos de cada par cromossômico. O número diplóide do cariótipo obtido dos cultivos celulares foi 2n = 12; estes se classificaram morfologicamente, de acordo com patrões previamente estabelecidos, assim: os pares I, II, IV e V foram metacêntricos, e o par III foi submetacéntrico. Por sua vez, o par sexual foi heteromórfico, sendo o cromossoma X me-tacêntrico e o cromossoma Y submetacêntrico. As bandas C foram positivas para todos os pares cromossômicos. Conclusões: Se estabeleceram as características citogenéticas de L. sericata, cepa Bogotá, Colômbia, relacionadas com número, forma, tamanho, posição do centrômero e regiões heterocromáticas dos cromossomas.

Succession pattern of cadaverous entomofauna in a semi-rural area of Bogotá, Colombia.

The main objective of this work was to examine the succession of insects colonizing three pig (Sus scrofa) cadavers in a semi-rural area of Bogotá. The 12kg pigs were shot and put into metallic mesh cages to allow access by insects. Arthropods were then sampled at different intervals depending on the corresponding stage of decomposition. In total 5981 arthropods were collected during decomposition, 3382 adults and 2599 immature stages, belonging to 10 orders and 27 families. Sarconesia magellanica and Compsomyiops verena (Diptera: Calliphoridae) were the first species to colonize the corpses. Egg masses and 1st stage Calliphoridae larvae were associated with the fresh stage of decomposition, 1st and 2nd stage larvae of Calliphoridae and Sarcophagidae during chromatic and emphysematous stages, immature Chrysomya albiceps (Diptera: Calliphoridae), Ophyra sp. (Diptera: Muscidae) and Oxellytrum discicolle (Coleoptera: Silphidae) during the colliquative stage and mainly Coleoptera during the skeletization phase (plus some adult Diptera). The data obtained in the present investigation could be used for the estimation of postmortem interval (PMI) in real cases when the conditions to which a cadaver has been exposed are similar to those recorded during this work.

Biocirugía: utilización de larvas de insectos necrófagos en la curación de heridas. La terapia larval / Biosurgery: use of larvae of necrophage insects in wound healing. The larval therapy

La terapia larval es una técnica que permite aplicar las larvas de algunas especies de dípteros sobre heridas crónicas o sobre focos de infección localizados, con la finalidad de restaurar el tejido afectado. La velocidad y la eficacia del tratamiento, sumadas a la casi nula pérdida de tejido sano durante el proceso de reparación tisular, han hecho de la terapia larval o biocirugía una alternativa viable para la curación de heridas asociadas con entidades como pie diabético, úlceras venosas, úlceras crónicas de la piel y quemaduras, así como de ciertos tipos de tumores beningos, abscesos y osteomielitis.Aunque la medicina moderna ha sido, en muchos casos, reticente a la aplicación de terapias de esta índole, eventos como la resistencia a los antibióticos y las alteraciones en el proceso de cicatrización en las heridas crónicas han permitido modificar la posición inicial de muchos médicos al respecto.