Banca de DEFESA: STEPHANNIE JANAINA MAIA DE SOUZA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : STEPHANNIE JANAINA MAIA DE SOUZA
DATE: 09/04/2021
TIME: 08:30
LOCAL: Vídeoconferência
TITLE:

Evaluation of the antiviral activity of red propolis from Alagoas against the Chikungunya virus in vitro

KEY WORDS:

Chikungunya virus, Red própolis, Antiviral activity.

PAGES: 64
BIG AREA: Ciências Biológicas
AREA: Microbiologia
SUBÁREA: Biologia e Fisiologia dos Microorganismos
SPECIALTY: Virologia
SUMMARY:

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 against CHIKV in vitro. First, CHIKV was isolated from serum 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 PVA was performed by high performance liquid chromatography (HPLC-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, 22.57 μg / mL and > 40,0 μg / mL was obtained for the hydroalcoholic extract (HE), hexane fraction (HF) and ethyl acetate fraction (EAF), respectively. For the antiviral activity assays, CHIKV adsorption was performed for 2h on Vero E6 cells, followed by treatment with different concentrations of PVA solutions for 72h and cell viability analysis 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 for the 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 plaque-forming units (PFU). As result, a reduction in the number of PFUs was detected [4,867 PFU / mL (EH), 12,767 PFU / mL (HF) and 8,867 PFU / mL 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 2h before CHIKV infection, simultaneously with infection (0h), or for different times post-infection (2h, 4h and 6h). Interestingly, the inhibitory activity was detected at 2h, 4h and 6h post- infection for both HE and HF and 2h and 4h after infection for EAF. To analyze the activity of the compounds detected by HPLC-DAD against the CHIKV proteins, a molecular Docking silica assay was performed, as a result of all the compounds detected above as targets of their activities, the CHIKV E3-E2-E1 glycoproteins complex. In conclusion, the data obtained in the present study demonstrate the promising antiviral activity of ARP against CHIKV in vitro, thus contributing to the development of new therapeutic agents for this arbovirus.

BANKING MEMBERS:
Externo ao Programa - 3182336 - EDEILDO FERREIRA DA SILVA JUNIOR
Interno - 1612086 - ENIO JOSE BASSI
Interno - 1488396 - TICIANO GOMES DO NASCIMENTO