Coxsackievirus B3 gains resistance to DENSpm via mutation in 2A protease. (A) Vero-E6 cells were left untreated or treated with 100 μM DENSpm for 16 h prior to infection with CVB3 at an MOI 0.1. Virus was collected at 24 hpi and used to inoculate the next passage. Viral titers were determined via plaque assay for the passages shown. (B) CVB3 passaged 10 times over Vero-E6 cells, either treated with 100 μM or untreated, were used to infect Vero cells treated with increasing doses of DENspm for 24 hpi. Viral titers were determined by plaque assay. (C) Partial 2A protease sequence of CVB3 compared to other enteroviruses. Red arrows indicate amino acid residues of the protease active site, and the black arrow indicates mutated amino acid residue of 2AS35 mutants. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 using Student’s t-test (n ≥ 3), comparing treated samples to untreated controls. Error bars represent ± 1 SEM.