The assay of protease activity of purified NS2B(H)-NS3(pro) was carried out using previously reported fluorogenic peptides from JEV conserved cleavage sites NS2B/NS3 (Pyr-RTKR-amc) and NS2A/NS2B (Dabcyl-PNKKRGWP-(EDANS)G) synthesized by A+Peptide (Pudong, Shanghai, China) [42 (link)]. Assays were conducted on 96-well black, flat bottom, tissue culture treated polystyrene microplates (Corning Life Sciences, MA, USA) in total reaction volume of 0.1 ml containing 0.5 mM NS2B(H)-NS3(pro) proteases, assay buffer (50mM Tris HCl, pH9.5, 20% glycerol) and substrate. For kinetic assessments, fluorescence measurements were recorded at 20 minutes interval for 520 minutes through Multimode plate reader (Bio Tek) at an excitation wavelength(λex) of 360nm and emission wavelength(λem) of 460 nm. Assay was carried out with three repeats for each group in two independent experiments at a constant temperature of 37°C. Inner filter effects of microplate reader were corrected as described in literature [46 (link)]. To determine the amount of AMC/fluorescence released, a standard curve was plotted with various value of kinetic data. No significant hydrolysis of peptide substrate was noted in dead NS2B(H)-NS3(pro) and/or groups without enzymes. Data was analyzed using GraphPad Prism version 7.00 for windows (GraphPad Software, La Jolla, California, USA)[47 , 48 (link)].