FIG 1.
Enterovirus 71 (EV-A71) recombination in RD cells is primarily replicative. (A) Cell-based EV-A71 recombination assay. C2 strain firefly luciferase-encoding subgenomic replicon (donor) and full-length EV-A71 C2-MP4 strain genome (acceptor) carrying a lethal deletion of the 3Dpol region were cotransfected in an equimolar ratio into RD cells. A fully functional virus genome can be produced via an RdRp template switch from donor to acceptor (indicated by dashed black arrow). (B) Only upon cotransfection can replication-competent virus be generated (PFU/ml ± standard deviations [SD]; n = 3) (C) Example sequences of plaque-purified recombinant virus from C2/C2 (left panel). Dashed arrows indicate predicted paths of viral RdRp upon template switching. Numbering refers to the position upon the acceptor templates. Lowercase, boldface nucleotides indicate the 5ʹ and 3ʹ boundaries of recombination. The underlined sequences indicate region of homology. (D) Nonreplicative recombination assay. IRES deletion of the C2 donor template inhibits translation. The acceptor template remains the same as in panel A. Viable virus will only be produced via a cell-mediated event. (E) Yield of recombinant virus (PFU/ml ± SD; n = 3) originating from transfection in equimolar ratio of replicative and nonreplicative partners. Statistical analyses were performed using an unpaired, two-tailed t test (***, P = 0.0001).