RESUMO
Resumen Introducción: la COVID-19 es una enfermedad aguda del tracto respiratorio causada por el coronavirus SARS-CoV-2. A pesar de que han surgido algunas opciones quimioprofilácticas, aún no existe un tratamiento efectivo contra esta enfermedad. Objetivo: investigar las propiedades antivirales de las nanopartículas de plata sintetizadas contra SARS-CoV-2 usando modelos in vitro. Metodología: en este estudio se sintetizaron AgNPs por medio de un método electroquímico. La caracterización de las AgNPs se realizó mediante técnicas físicoquímicas (ICP-OES, espectroscopía UV-Vis, y microscopía electrónica de transmisión). El efecto citotóxico y la actividad antiviral de las AgNPs contra SARS-CoV-2 se evaluó in vitro en células Vero E6. La viabilidad celular se evaluó en presencia de diluciones seriadas de AgNPs mediante el ensayo de MTT. Adicionalmente, el efecto antiviral contra el SARS-CoV-2 fue evaluado por ensayo de placa en células Vero E6 bajo la estrategia prepostratamiento. El diámetro de las AgNPs estuvo entre 2,6 y 30 nm. El tamaño promedio obtenido por los métodos electroquímicos fue de 6,2 nm. Resultados: el efecto citotóxico fue observado a concentraciones por encima de 0,07 ppm. Cuando se empleó la estrategia pre-postratamiento con las AgNPs se observó una reducción significativa del título de SARS-CoV-2 con porcentajes de inhibición del 96,5 %, 64,13 % y 74,72 % en las concentraciones de 0,03, 0,017 y 0,008 ppm, respectivamente. Conclusión: nuestros resultados sugieren que las AgNPs podrían reducir la replicación del SARS-CoV-2, con un bajo efecto citotóxico. Sin embargo, se requieren estudios adicionales tanto in vitro como in vivo para definir su potencial terapéutico en humanos.
Abstract Introduction: COVID-19 is an acute respiratory tract disease caused by the emerging coronavirus SARS-CoV-2. Although several options for chemoprophylaxis are under development, effective treatment for COVID-19 is not yet available. Objective: To investigate the antiviral properties of synthesized silver nanoparticles (AgNPs) against SARS-CoV-2 using in vitro models. Materials and methods: This work synthesized AgNPs using an electrochemical method and characterized them using physico-chemical techniques (ICP-OES, ultraviolet-visible spectroscopy, and transmission electron microscopy). AgNPs with diameter sizes ranging between 2.6 to 30 nm and an average size of 6.2 nm were obtained by the electrochemical method. The cytotoxic effect and the antiviral activity of prepared AgNPs against SARS-CoV-2 were evaluated in vitro using Vero E6 cells. Cell viability was evaluated by MTT assay in the presence of serial dilutions of AgNPs. The antiviral effect of AgNPs was evaluated before and after the infection of Vero E6 cells by plaque assay. Results: Cytotoxic effect was observed at concentrations above 0.07 ppm. AgNPs exhibit a significant reduction of SARS-CoV-2 viral titer after a prepost treatment strategy with inhibition of 96.5%, 64.13%, and 74.72% at 0.03, 0.017, and 0.008 ppm, respectively. Conclusion: Our results suggest that AgNPs could reduce SARS-CoV-2 replication with a low cytotoxic effect. Still, additional in vitro and in vivo studies are required to define its potential therapeutic application in humans.
RESUMO
Ehrlichia canis is the etiologic agent of a highly prevalent tick-borne disease, canine monocytic ehrlichiosis (CME). Four defined E. canis genotypes based on the trp36 gene sequences have been reported, three of them identified in North or South America. The diversity of E. canis has been investigated using genetic and serologic approaches based on distinct 36â¯kDa tandem repeat protein (trp36) gene sequences that have been reported. The main objectives of this study were to determine the prevalence of E. canis infection in dogs from Medellín, Colombia by PCR and determine the E. canis diversity using molecular and serologic approaches. Blood was collected from dogs (nâ¯=â¯300) with clinical signs of CME for PCR detection of E. canis 16S rRNA, dsb and trp36 DNA. Phylogenetic analysis of trp36 gene sequences was performed using MEGA. A serological evaluation was performed using immunofluorescence microscopy and ELISA with species-specific peptides from E. canis TRP19 and TRP36 (3 genotypes) and E. chaffeensis (TRP32). E. canis DNA (16S rRNA and/or dsb) was detected in 18 % (53/300) of dogs by PCR amplification. The trp36 gene was amplified and sequenced from 35/53 16S rRNA/dsb PCR positive samples revealing three genotypes: United States (US; nâ¯=â¯21), Costa Rica (CR; nâ¯=â¯11), and Brazil (BR; nâ¯=â¯3). Most dogs (33/35) with detectable trp36 DNA had anti-E. canis TRP19 and TRP36 peptide antibodies that corresponded to the genotype detected by PCR. Dogs that had antibodies to the TRP19 peptide (82/300; 38 %), also had antibodies to one or more genotype-specific TRP36 peptides. Based on TRP36 serology, the dogs exhibited highest frequency of infection with the US genogroup (USâ¯=â¯26), followed by the CR genogroup (CRâ¯=â¯19) and the BR genogroup (BRâ¯=â¯11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CRâ¯=â¯8, BR/CRâ¯=â¯7, US/CRâ¯=â¯6 and US/BRâ¯=â¯5) suggesting coinfection or multiple sequential infections with different genotypes. Colombian dogs did not have antibodies to E. chaffeensis as determined by a TRP32 species-specific ELISA. Our results demonstrate the presence of three previously defined genotypes in North and South America in Colombian dogs (US, BR, CR). These results also demonstrate that TRP19 and TRP36 serology can provide valuable information regarding E. canis exposure and the potential genotype(s) involved in infection.