The CRE-REP assay to analyse recombination in enteroviruses.

<p>A) Schematic diagram of enterovirus recombination partners and products. The upper panel shows a diagram of a genome (dark shading) bearing a defective CRE indicated as a broken line with a superimposed X in the 2C-coding region and a luciferase-encoding replicon (light shading). Following co-transfection into permissive cells (indicated by an arrow), a replication competent recombinant genome may be recovered of the generic structure shown, consisting of the 5′ part of the genome derived from the CRE-defective parent (the recipient) and the 3′ part of the genome from the luciferase-encoding replicon (the donor). The graduated shading between the 3′ end of the VP1-coding region and the 2C CRE indicates the area within which recombination must occur to produce a functional genome. B) Schematic diagram of the recombinant production strategy. RNA generated <i>in vitro</i> was co-transfected into cells permissive for virus replication but not susceptible to infection. After 24–48 hours, supernatant virus was recovered, cloned by limit dilution and subjected to further analysis. C) Xylene cyanol-stained HeLa cell monolayers infected with supernatant from murine L929 cells transfected with RNA derived from the plasmids indicated. Guanidine hydrochloride (GuHCl; 4 mM) added where indicated to HeLa cells. D) Comparison of inter- and intra-serotypic recombination following independent co-transfection of L929 murine fibroblasts with RNA generated from poliovirus type 3 (PV3/PV3) or poliovirus type 1 and type 3 (PV1/PV3) cDNAs. The error bars indicate the standard deviation of three independent assays with significance determine by chi square test (<i>p</i> = 0.01).