Objective: To draw a representing consensus sequence of each DENV serotype, align all four consensus sequences to draw a global consensus sequence and also study the highly conserved
residues. Methods: A total of 376 DENV NS3 sequences, belonging to four serotypes, reported from all over the world were aligned to develop global consensus sequence. Results: The active site residues Met343, Thr366, which are involved in nuclear localization and also interact with the NS3 viral, are highly conserved among all the DENV serotypes. Cys450, Gly466 and Ala468, Arg482 are highly conserved in all the serotypes. Structural zinc (Zn1) sited consist of Cys- 446, Cys-449, His-441, and the carboxylate group of Glu-437. This pocket is also found near the functionally important residues Ser-710 and Arg-729, which bind to the incoming rNTP. Meth530, Thr543 Asp597, Glu616 and Arg659, Pro671 are structurally conserved in all serotypes. Identification of four out of six conserved sequence motifs accountable for NTP binding and GDD catalytic active site, located in the palm domain. Leu766, Ala776 residues have high conservancy in all serotypes are observed in consensus sequence analysis. The thumb domain also has Zinc binding site (Zn2) and is synchronized by His-712, His-714, Cys-728 of motif E, and Cys-847. Pharmacological blockage of cavity B could potentially lead to suppression of initiation of the viral RNA synthesis and/or inhibition of NS3/NS5 interaction. Thirteen different peptides from the highly conserved regions of DENV NS5 protein were drawn which can be used to develop peptidic inhibitors. Conclusions: In spite of a high mutation rate in DENV, the residues which are present in the Nuclear localization signal (NLS), Di-valet ion binding sites, NTP binding, GDD catalytic active site, Thumb domain, priming loop are highly conserved. These are target sites for the development of antiviral agents or peptide vaccines.