The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the "super" deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.