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. 2014 Dec 15;1(2):98–109. doi: 10.1021/id500022h

Figure 1.

Figure 1

High-throughput screen design and execution: recombinant influenza virus and high-throughput screen design and results. (a) Schematic of the recombinant influenza A/WSN/33 virus expressing Renilla luciferase. The Renilla luciferase open reading frame was inserted in the reverse orientation and complementary sense between the 3′ and 5′ packaging sequences of the HA segment, which contains the viral promoter and ensures correct packaging of the recombinant segment. Due to the lack of HA ORF, this WSN-Renilla virus must be grown in an HA-complementing cell line. Upon infection, the influenza virus polymerase recognizes the promoter and the reporter gene is transcribed and expressed. (b) MDCK-HA cells were plated in 1536-well plates and infected with WSN-Renilla virus (MOI = 0.05). Compounds were added 120 min prior to infection, and expression of Renilla luciferase was assayed 30 h later. A 50% reduction in luminescence signal was employed as a cutoff. (c) Results from the HTS of 919,960 compounds indicating the number of primary hits, the hits confirmed in dose–response, and the selection of hits for revalidation.