Secondary screen for inhibition of human norovirus Sydney 2012 RdRp activity. (A) The inhibitory effects of 26 compounds were examined against norovirus RdRp activity using a fluorescent activity assay at 10 µM. Five compounds that exhibited more than 50% inhibition (red bars) were chosen for further assessment with a counter-screen gel-shift RdRp assay. The positive control (blue bar) is the norovirus NNI RdRp inhibitor NIC0214. The mean values of triplicate datasets with standard deviations are shown. (B) A counter-screen-gel-shift assay was used to confirm norovirus RdRp inhibitory activity of the five compounds identified in panel (A). PE44-NoV RNA templates (32 nucleotides) were extended (44 nucleotides) by the RdRp in the absence of any test compounds (0.5% DMSO [vol/vol] negative control) or with test compounds at a fixed concentration of 100 µM. The nucleoside analogue 3′-deoxyguanosine-5′-triphosphate (3′dGTP) was used as a positive control (10 µM) to demonstrate complete inhibition, and no RdRp was used as a negative control. Full-length gels are presented in Supplementary Fig. S3.