Arboviruses such as Dengue, yellow fever, West Nile, and Zika are flaviviruses vector-borne RNA viruses transmitted biologically among vertebrate hosts by blood-taking vectors. Many flaviviruses are associated with neurological, viscerotropic, and hemorrhagic diseases, posing significant health and socioeconomic concerns as they adapt to new environments. Licensed drugs against them are currently unavailable, so searching for effective antiviral molecules is still necessary. Epigallocatechin molecules, a green tea polyphenol, have shown great virucidal potential against flaviviruses, including DENV, WNV, and ZIKV. The interaction of EGCG with the viral envelope protein and viral protease, mainly identified by computational studies, describes the interaction of these molecules with viral proteins; however, how the viral NS2B/NS3 protease interacts with epigallocatechin molecules is not yet fully deciphered. Consequently, we tested the antiviral potential of two epigallocatechin molecules (EGC and EGCG) and their derivative (AcEGCG) against DENV, YFV, WNV, and ZIKV NS2B/NS3 protease. Thus, we assayed the effect of the molecules and found that a mixture of the molecules EGC (competitive) and EGCG (noncompetitive) inhibited the virus protease of YFV, WNV, and ZIKV more effectively with IC₅₀ values of 1.17 ± 0.2 µM, 0.58 ± 0.07 µM, and 0.57 ± 0.05 µM, respectively. As these molecules fundamentally differ in their inhibitory mode and chemical structure, our finding may open a new line for developing more effective allosteric/active site inhibitors to combat flaviviruses infection.

Chikungunya virus (CHIKV) is an emerging arbovirus whose replication relies on the multifunctional nonstructural protein 2 (nsP2), particularly its viral protease (nsP2 ^pro ), which is essential for polyprotein processing. In this study, we investigated how interactions with nucleic acids influence nsP2 ^pro activity. Using high-throughput sequencing–fluorescent ligand interaction profiling, we identified specific single-stranded DNA aptamers that enhanced nsP2 ^pro activity. Additionally, both random single-stranded DNA and single-stranded RNA were found to stimulate protease activity, whereas double-stranded DNA showed no such effect. Circular dichroism spectroscopy and secondary structure predictions confirmed that the identified aptamers adopt stable folded conformations. Similarly, structured RNA sequences were also capable of promoting protease activity. The observed stimulatory effect depended on the nucleic acid strand type, length, and buffer conditions, suggesting the involvement of electrostatic interactions. Molecular docking analyses further let us assume that these nucleic acids interact specifically with the nsP2 ^pro methyltransferase domain. Our findings provide novel insights into the regulation of nsP2 ^pro , enhancing our understanding of CHIKV replication mechanisms, and may guide future antiviral development strategies.