Technical Abstract: Foot-and-mouth disease virus (FMDV) initiates infection in vitro via recognition of at least four cell-surface integrin molecules avb1, avb3, avb6 or avb8 through the interaction of a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the GH loop of VP1. In this work, soluble integrin resistant (SIR) mutants were selected upon passages of O1 Campos or A24 Cruzeiro on bovine kidney (LFBK) cells in the presence of sub-neutralizing levels of soluble avb6 (ss avb6) heterodimers. SIR FMDV mutants were isolated and characterized by: sequence, sensitivity to neutralization by ss avb6, ability to utilize avb6 for attachment, and mode of uptake. Regardless of the serotype, all SIR mutants maintained avb6 receptor affinity for entry and either acquired the ability to be internalized by cells expressing heparan sulfate proteoglycan or a still unknown third receptor. Two classes of A24 Cruzeiro SIR mutants were selected; a high-resistance class, which resisted neutralization by 1000-5000 fold, and a low-resistant class, which demonstrated a 300-500 fold increased resistance to neutralization by ss avb6 (20 mg/ml) compared to wild-type virus. These A-type mutants showed an alteration in the receptor-binding site (RGD > RDD) and/or in additional downstream VP1 sequences. These findings demonstrate the plasticity of FMDV-host cell interactions, allowing the virus to rapidly evolve resistance to soluble receptor prophylactic measures.