Schematic representation of the IBV genome organization depicting all the modifications tested for IBV attenuation and their respective outcomes. Site-directed mutagenesis and inverse PCR were used to incorporate the attenuation modifications into the PB1 (E391, G580, A660, and HA tag) and PB2 (S267, F359, and Y406) gene segments. The PB1 E391 modification was unstable. PB2 S267 was deleterious for virus rescue, while the PB2 F359 and Y406 substitutions increased IBV virulence. The G580, A660, and HA tag modifications in the PB1 segment were stable and sufficient to confer attenuation (PB1 att).