Elucidation of the antiviral mechanism of natural therapeutic molecules Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue virus

Arthropod-borne viruses of the alphavirus and flavivirus genera are human pathogens of significant concern, and currently, no specific antiviral treatment is available for these viruses. In this study, the antiviral mechanisms of natural small molecules against Dengue virus (DENV) and Chikungunya virus (CHIKV) have been investigated. Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) showed depletion of polyamine levels in Vero cells as demonstrated by thin-layer chromatography (TLC). As polyamines are known to play a role in viral replication and transcription, HC and CAPE were expected to inhibit virus replication by reducing polyamine levels. To test this hypothesis, HC and CAPE were evaluated for antiviral activities using a cell-based virus yield assay by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), plaque reduction assay, and immunofluorescence assay (IFA). HC and CAPE displayed potent inhibition with EC50 of 463 nM and 0.417 nM for CHIKV and 8.5 µM and 1.15 µM for DENV, respectively. Interestingly, however, the addition of exogenous polyamines did not completely rescue the virus titer in both CHIKV and DENV infected cells and this indicated additional antiviral mechanisms for HC and CAPE. Further, in silico analysis indicated that HC and CAPE directly target the viral methyltransferases (MTase) of CHIKV and DENV. A high throughput ELISA-based assay that quantifies m7GMP-nsP1 adduct was employed to validate inhibition of CHIKV nsP1 MTase and IC50 was calculated to be 0.009 µM and 0.08 µM for CAPE and HC respectively. Altogether, the identification of natural small molecules as antivirals opens the door for the development of antiviral therapies for the treatment of CHIKV and DENV infections.