Metodología de pesquisa primaria de actividad antidengue a partir de productos naturales / Methodology for primary screening of antidengue activity based on natural products

Introducción: El virus dengue, transmitido por mosquitos del género Aedes ha reemergido en los últimos años produciendo la enfermedad transmitida por artrópodos con mayor prevalencia en humanos, y no existe una terapia antiviral específica ni vacunas, para su tratamiento y prevención. Ello ha motivado la búsqueda de productos y compuestos naturales con actividad antiviral, lo cual trae consigo la necesidad de establecer un sistema de evaluación de productos naturales y sintéticos mediante una metodología de pesquisa rápida in vitro. Objetivo: Proponer un sistema de pesquisa primaria de actividad antiviral contra el virus dengue basado en células. Métodos: Se utilizaron como fuentes primarias de información trabajos publicados en revistas nacionales e internacionales registradas en las bases de datos SciELO o PubMed. Los ejemplos seleccionados en las figuras y tabla proceden de las publicaciones conjuntas del Grupo de Virología de la Facultad de Biología de la Universidad de La Habana y del Laboratorio de Arbovirus del Instituto de Medicina Tropical Pedro Kourí. Información, análisis y síntesis: Se presentan las principales metodologías basadas en células, y se enfatiza en aquellas asumidas por nuestro grupo (evaluación de la citotoxicidad y el ensayo primario de actividad antiviral). Se muestra el algoritmo de evaluación de un producto. La metodología descrita ha permitido poner en marcha un programa de búsqueda de fármacos antidengue, teniendo en cuenta los criterios de la evaluación de la eficacia antiviral y la toxicidad, para realizar un estudio posterior de mecanismo de acción de los diferentes compuestos o productos evaluados.

Introduction: Dengue virus, transmitted by Aedes specie mosquitos, has re-emerged in the last years causing the arthropod-borne disease with higher prevalence in humans, to which there is no specific antiviral therapy or vaccine for its treatment and prevention. This has motivated the search for natural-based products with antiviral activity, which implies the need to establish an evaluation system of natural and synthetic products through a rapid in vitro screening methodology. Objective: To propose a primary screening cell-based antiviral activity system against dengue virus. Methods: Papers published in national and international journals indexed in SciELO or PubMed were used as primary sources of information. The examples selected in the figures and table were retrieved from the joint publications of the Virology Group of the School of Biology of the University of Havana and the Arbovirus Laboratory of the Institute of Tropical Medicine Pedro Kourí. Information, Analysis and Synthesis: The main cell-based methodologies are presented, with emphasis on those assumed by our research group (evaluation of cytotoxicity and the primary antiviral activity assay). The algorithm for product evaluation is presented. The methodology described has allowed initiating a search program for antidengue drugs, taking into account the criteria for evaluating antiviral efficacy and toxicity, in order to carry out a subsequent study on the mechanisms of action of the different compounds or products evaluated.

In vitro Evaluation of Anti-Chikungunya Virus Activities of Tualang Honey

@#Chikungunya is an infection caused by chikungunya virus (CHIKV). Although chikungunya has affected many countries in recent times, specific treatment or licensed vaccine are nonexistent. In this study the potential antiviral properties of Tualang honey against in vitro CHIKV infection was evaluated. Cytotoxic test was performed using the XTT Cell Viability assay to determine maximum non-toxic dose (MNTD) in Vero cells. Using plaque assay, the potential antiviral activities of Tualang honey at various non-toxic concentrations and treatment regimens were evaluated. Tualang honey demonstrated virucidal effect with maximum inhibition CHIKV observed was 99.71% (p< 0.05). Tualang honey also had a prophylactic property by conferring protection to Vero cells during pre-treatment assay, resulting in up to 98.22% reduction of CHIKV replication under certain treatment regimen. Furthermore, Tualang honey exhibited anti-viral activities, with as much as 94.87% inhibition following post-treatment assay of Tualang honey in CHIKV-infected Vero cells. Additionally, Tualang honey also affected viral entry up to 82.21% after 48 hours of infection. These results suggest that Tualang honey has wide anti-CHIKV activities in Vero cells and exerts its effect through different mechanisms although these need to be further validated in other cells or model of CHIKV infection.

Steroidal saponins from the roots of Solanum sisymbriifolium Lam. (Solanaceae) have inhibitory activity against dengue virus and yellow fever virus

Dengue is the most important arthropod-borne viral disease worldwide. Infection with any of the four dengue virus (DENV) serotypes can be asymptomatic or lead to disease with clinical symptoms ranging from undifferentiated and self-limiting fever to severe dengue disease, which can be fatal in some cases. Currently, no specific antiviral compound is available for treating DENV. The aim of this study was to identify compounds in plants from Paraguayan folk medicine with inhibitory effects against DENV. We found high virucidal activity (50% maximal effective concentration (EC50) value of 24.97 µg/mL) against DENV-2 in the ethanolic extract of the roots of Solanum sisymbriifolium Lam. (Solanaceae) without an evident cytotoxic effect on Vero E6 cells. Three saponins isolated from the root extract showed virucidal effects (EC50 values ranging from 24.9 to 35.1 µg/mL) against DENV-2. Additionally, the saponins showed inhibitory activity against yellow fever virus (EC50 values ranging from 126 to 302.6 µg/mL), the prototype virus of the Flavivirus genus, suggesting that they may also be effective against other members of this genus. Consequently, these saponins may be lead compounds for the development of antiviral agents.

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)

Transformation and standardization of the inactivation test method for virus suspension in biosafety laboratory / 中国比较医学杂志

Chemical disinfectants are generally used for virus inactivation and environment disinfection in biosafety laboratory, and the efficacy and evaluation of the disinfection are critical to ensure the laboratory biosafety.However, there is a current lack of applied standard to evaluate the virucidal efficacy of chemical disinfectants in our country.In this paper, a European Union standard“Method and Requirements of Virucidal Quantitative Suspension Test Method for Chemical Disinfectants Used in Human Medicine” was analyzed and a standard transformation scheme has been proposed.It is suggested that the model viruses should be increased from 3 to 6, including the surrogate viruses to substitute highly pathogenic viruses, and that the method to remove the residual chemical disinfectant and the calculation of 95%confidence interval should be incorporated into the standard.The suggestion will improve the scientific and operational standards related to disinfection and sterilization in biosafety laboratory in China.

Acción virucida directa sobre el virus influenza de un extracto de Punica granatum L (granada) / Direct virucidal action against influenza virus of an extract of Punica granatum L (granada)

INTRODUCCIÓN: la especie vegetal Punica granatum L. se conoce en Cuba como granada. A esta planta se le atribuye acción anticatarral en la medicina tradicional, entre otras propiedades. En estudios previos realizados al extracto hidroalcohólico liofilizado preparado con el fruto de esta planta, se informaron la presencia de numerosos componentes químicos, la mayoría flavonoides. Estos compuestos, unidos a la variedad de metabolitos secundarios detectados, le confieren a este liofilizado la posibilidad de poseer acción antiinfluenza, tal como ha sido demostrado en estudios previos del mismo. OBJETIVO: evaluar la acción virucida directa del extracto hidroalcohólico liofilizado de P. granatum frente a la cepa Influenza A/Mississippi/1/85 (H3N2), con diferentes variantes de tratamiento. MÉTODOS: las variantes de tratamiento directo se realizaron en 27 combinaciones de las variables; tiempo de contacto de: 60, 30 y 15 minutos; concentración del extracto de: 1000, 200 y 125 µg/mL y temperatura de incubación de la mezcla extracto-virus: 4, 25 y 37 0C. La presencia o no de la hemaglutinina del virus influenza en cada una de las muestras tratadas, se midió por la titulación de la Inhibición de la Hemaglutinación (IHA) y el título infectivo viral (DIE50). RESULTADOS: la concentración mínima efectiva para ejercer el extracto liofilizado, la acción virucida directa, in vitro, fue 125 µg/mL, a los 15 minutos. La acción virucida directa de éste extracto estuvo siempre presente en las diferentes temperaturas y tiempos de exposición ensayados. CONCLUSIONES: la variante de ensayo que utilizó 125 µg/mL, con independencia del tiempo y la temperatura de tratamiento, resultó suficiente efectiva para reducir la presencia de hemaglutinina del virus influenza.

INTRODUCTION: Punica granatum L. is known in Cuba as grenade. In traditional medicine, among other properties, antiflu action is attributed to this plant. In previous studies with the hydroalcoholic lyophilized extract, prepared with the fruit of this specie, several chemical compounds were reported, specially the presence of flavonoids. The variety of secondary metabolites detected for this lyophilized extract, added to a flavonoid fraction confers a high possibility of anti-influenza activity, as has been demonstrated in previous studies. OBJECTIVE: to evaluate the virucidal activity of the hydroalcoholic lyophilized extract of P. granatum against Influenza A/Mississippi/1/85 (H3N2), with different schemes of treatments. METHODS: the different schemes of treatments included 27 variants of the variables such as contact time of 60, 30 and 15 minutes; extract concentration of 1000, 200 and 125 µg/mL; an incubation temperature of the mix extract-virus of 4, 25 and 37 0C; and the corresponding replicas. The presence or not of hemaglutinin of influenza virus in each one of the treated samples, was measured by titration of Hemaglutination Inhibition (HAI) and the Embryo Infection Doses (EID50). RESULTS: the minimal effective concentration to show in the hydroalcoholic lyophilized extract the direct virucidal action in vitro was 125 µg/mL, in 15 minutes of contact. This virucidal action was always present in the different assessed temperature and time exposures. CONCLUSIONS: the treatment of 125 µg/mL, regardless of time and temperature of treatment, was effective enough to reduce the presence of hemaglutinin of the influenza virus.

In vitro antiviral activity of seeds from Guettarda angelica against avian viruses.

The aim of this study was to evaluate the inhibition of the aqueous extract of seeds (AEs) from Guettarda angelica on cell infection by two avian RNA viruses: avian reovirus (ARV) and metapneumovirus (AMPV). The cytotoxic and antiviral activities were evaluated by MTT assay to determine the 50% cytotoxic (CC50) and inhibitory concentrations (IC50). The selectivity index (SI=CC50/IC50) also carried out. AEs exhibited antiviral activity only against ARV presenting IC50 of 23.59μg/mL. This inhibition was not due to any cytotoxic effect of AEs since the CC50 on Vero cells was of 400.60μg/mL and its SI was of 17.00; this extract also showed a virucidal effect on ARV. Previous studies also demonstrated antiviral activity of AEs extract against three animal herpesviruses. Thus, the seeds from G. angelica showing antiviral activity against DNA and RNA viruses, enveloped and non-enveloped, could be promising source of new antiviral agents encouraging its fractionation to isolate the active compound.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50 percent reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Virucidal activity presence in Trichilia glabra leaves / Presencia de actividad antiviral en hojas de Trichilia glabra

Different immunomodulatory activities present in Trichilia glabra (TG) leaf extracts have already been described. Particularly, chloroform-methanol extracts were responsible for an in-vivo anti-inflammatory effect. The effect of such extracts on the infectivity of enveloped and naked viruses were investigated. Methanolic fraction extracts were active against herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV), while no activity against poliovirus type 3 was observed. VSV was slightly more affected than HSV-1: 2.8 log10 reduction in VSV titer against 2.4 log10reduction in HSV-1 titer when 0.25 mg/ml F2 fraction was tested and a reduction of 2.7 log10in VSV virus titer and of 1.5 log10in HSV-1 virus titer was observed when 0.25 mg/ml F3 fraction was tested. Results obtained in this work suggest a potential pharmaceutical use of TG extract components.

Previamente se han descripto distintas actividades inmunomoduladoras, presentes en extractos de hojas de Trichilia glabra (TG). En particular, se ha demostrado una actividad antiinflamatoria presente en extractos metanólicos. En este trabajo se investigó la actividad virucida de dichos extractos sobre virus envueltos y desnudos. Distintos extractos metanólicos han inactivado en forma moderada los virus herpes simplex tipo 1 (HSV-1) y el virus de la estomatitis vesicular (VSV), mientras no evidenciaron actividad sobre poliovirus tipo 3. VSV resultó algo mas afectado que HSV-1: se observó una reducción en el título viral de 2,8 log10para VSV y de 2,4 log10para HSV-1 cuando se uso una concentración de 0,25 mg/ml de la fracción F2 y una reducción de 2,7 log10para VSV y de 1,5 log 10para HSV-1 cuando se usó una concentración de 0,25 mg/ml de la fracción F3. Los resultados obtenidos en este trabajo, sugieren un potencial uso farmacéutico de los componentes presentes en los extractos de TG.