EFFECT OF NATURAL PRODUCTS DERIVATIVE FROM SCHINUS TEREBINTHIFOLIUS AND PUNICA GRANATUM ON ARBOVIRUS REPLICATION

MENESES, M.D.F.; SALLES, T.S; CAMPOS R.DE M.; MALNERO, C.M; BERGMANN I.E.; MALIRAT V,; KUSTER RM; SILVA MRS; FERREIRA, D.F.

Buenos Aires

Congreso; XI Congreso Argentino de Virología (CAV 2015), II Congreso Latinoamericano de Virología; 2015

Sociedad Argetina de Virología

Many human diseases are caused by arboviruses, the symptoms can range from mild febrile illness to encephalitis and death. Recently, American continent has been facing the burden of introduction and re-introduction of arboviruses such as Dengue virus (DENV), Chikungunya virus, Japanese encephalitis virus, and West Nile virus in urban areas. Most of the pathogenic arbovirus species belong mainly to 3 families: Togaviridae, Flaviviridae, and Bunyaviridae. Mayaro virus (MAYV) belongs to the genus Alphavirus, Togaviridae family, which causes a disease presenting symptoms similar to the classic dengue fever and was used as Alphavirus prototype. The Brazilian popular medicine explores the anti-inflamatory properties of substances extracted from the plants Schinus terebinthifolius (mastic) and Punica granatum (Pomegranate). In this work we present studies on the antiviral effect of these substances against MAYV and DENV. The toxicity of these substances in Vero cells was tested by the incorporation of neutral red after 24h of treatment. The CC50 of each substance was determined: 242, 315, 102 and 5000 ìg/ml in acetate (mastic), flavonoids 1 and 2 (mastic), oil of Mastic, respectively, and 590 and 442 ìg/ml for Crude Extract and Acetate of Pomegranate, respectively. Afterwards, tests for evaluation of antiviral effect were carried out. Cells were infected for 1 h with MAYV using a multiplicity of infection of 0.1, and treated for 24h with increasing concentrations of the substances. Viral yield, quantified by TCID50 method, demonstrated antiviral activity. The IC50 of each substance was determined by a dose response graph, and values obtained were: 4,3; 4,5; 14 and 830 ìg/ml in acetate mastic, flavonoids 1 and 2 (mastic), oil of Mastic, respectively, and 12 and 30 ìg/ml for Crude Extract and Acetate of Pomegranate, respectively. The selectivity index (ratio CC50/IC50), was determined rendering values > 7 for all substances. We also tested the substances for virucide properties, adsorption impairment, by means of pretreatment, immunofluorescence and transmission electron microscopy (TEM). Our results showed more than 95% virucidal effect for the mastic acetate and flavonoids partitions and for the Crude Extract of pomegranate. Mastic oil 22%, and pomegranate acetate did not present virucidal effect. We observed similar inhibition obtained in the virucidal test in immunofluorescence images. Damage in viral particles treated with the substances was also observed in the TEM images obtained. The proteomic analysis of the infection of MAYV showed differences in the expression of secreted proteins during infection and treatment with flavonoid 1. We conclude that some of the partitions in our substances act directly on the virus particle. These results will be compared to the activity of these substances on DENV, of the Flavivirus genus belonging to Flaviviridae family. of these substances against MAYV and DENV. The toxicity of these substances in Vero cells was tested by the incorporation of neutral red after 24h of treatment. The CC50 of each substance was determined: 242, 315, 102 and 5000 ìg/ml in acetate (mastic), flavonoids 1 and 2 (mastic), oil of Mastic, respectively, and 590 and 442 ìg/ml for Crude Extract and Acetate of Pomegranate, respectively. Afterwards, tests for evaluation of antiviral effect were carried out. Cells were infected for 1 h with MAYV using a multiplicity of infection of 0.1, and treated for 24h with increasing concentrations of the substances. Viral yield, quantified by TCID50 method, demonstrated antiviral activity. The IC50 of each substance was determined by a dose response graph, and values obtained were: 4,3; 4,5; 14 and 830 ìg/ml in acetate mastic, flavonoids 1 and 2 (mastic), oil of Mastic, respectively, and 12 and 30 ìg/ml for Crude Extract and Acetate of Pomegranate, respectively. The selectivity index (ratio CC50/IC50), was determined rendering values > 7 for all substances. We also tested the substances for virucide properties, adsorption impairment, by means of pretreatment, immunofluorescence and transmission electron microscopy (TEM). Our results showed more than 95% virucidal effect for the mastic acetate and flavonoids partitions and for the Crude Extract of pomegranate. Mastic oil 22%, and pomegranate acetate did not present virucidal effect. We observed similar inhibition obtained in the virucidal test in immunofluorescence images. Damage in viral particles treated with the substances was also observed in the TEM images obtained. The proteomic analysis of the infection of MAYV showed differences in the expression of secreted proteins during infection and treatment with flavonoid 1. We conclude that some of the partitions in our substances act directly on the virus particle. These results will be compared to the activity of these substances on DENV, of the Flavivirus genus belonging to Flaviviridae family.