Avaliação da atividade antiviral da própolis vermelha de Alagoas contra o vírus Chikungunya in vitro

Abstract

Chikungunya virus (CHIKV) causes Chikungunya fever disease that can progress to the chronic phase characterized by persistent arthralgia. There are currently no antivirals or licensed vaccines against this virus. Alagoas Red Propolis (ARP) is a natural resin that bees produce by mixing saliva and exudate from plants that have antimicrobial activities. Therefore, our objective was the chemical characterization and evaluate the antiviral activity of ARP and its hexanic (HF) and ethyl acetate fractions (EAF) against CHIKV in vitro. First, CHIKV was isolated from serum of infectedpatients in the acute viremia phase of the disease, replicated in Vero E6 cells and confirmed by RT-PCR using primers for the E1 protein genomic region (882 bp). DNA sequencing and phylogenetic analysis (MEGA7 software) confirmed East/ Central/ South African (ECSA) as the CHIKV genotype. The chemical characterization of ARP was performed by ultra performance liquid chromatography (UPLC-DAD) and the presence of the liquiritigenin, pinobanksin, daidzein, formononetin, biochanin A and isoliquiritigenin markers were detected. The ARP cytotoxicity in Vero E6 cells was evaluated by MTT colorimetric assay and CC50 values of 200.1 μg / mL, 22.6 μg / mL and > 40.0 μg / mL was obtained for the hydroalcoholic extract (HE), HF and EAF, respectively. For the antiviral activity assays, CHIKV adsorption was performed for 2h on Vero E6 cells, followed by treatment with different concentrations of ARP solutions for 72h and cell viability analysed by MTT assay. As result, a promising antiviral activity was detected and the IC50 values were 22.80 μg/mL, 4.24 μg/mL and 5.34 μg/mL forthe HE, HF and EAF, respectively. In addition, high selectivity indices (SI) were obtained for the compounds (HE = 8.7, HF = 5.3 and EAF > 7.5). In order to confirm the antiviral activity, virus intracellular labeling was performed and the percentage of positive cells was detected by flow cytometry 48h after infection. As result, all tested ARP solutions showed a reduction effect higher than 50% on the percentage of infected cells. In the inactivation assay, CHIKV suspensions were first incubated with ARP for 2h, diluted and incubated for 48h, followed by the detection of the plaqueforming units (PFU), and a reduction in the number of PFUs was detected [4,867 PFU / mL (HE), 12,767 PFU / mL (HF) and 8,867 PFU / mL (EAF) vs. 31,000 PFU / mL untreated viral control]. In addition, to assess which stages of viral infection the compounds are acting on, cells were treated with ARP and their fractions for 2 h before CHIKV infection, simultaneously with infection (0h), or for different times post-infection (2 h, 4 h and 6 h). The inhibitory activity was detected 2h, 4h and 6h after infection at HE and HF assays, and 2h and 4h after infection at EAF assay. To analyze the activity of the compounds detected by UPLC-DAD against CHIKV mature envelope proteins, an in silico study of molecular docking was carried out. All detected compounds showed to target the glycoproteins of E3-E2-E1 CHIKV complex. Thus, the data obtained in the present study demonstrate the promising antiviral activity of ARP against CHIKV in vitro, contributing to the development of new therapeutic agents for this arbovirus