Evaluation of mutations in nsP2 and E2. The plasmid prSAV3 was modified to express either serine or proline in E2206 and either alanine or aspartic acid in nsP2197. (A) Infectious viruses were recovered from all plasmids after transfection into BF2 cells. The variants expressing the nsP2197A produced CPE in CHSE cells, while viruses expressing nsP2197 D did not. The E2206 mutation did not appear to affect CPE. Representative images of CPE are shown for each mutant. (B) The E2206 mutation did not affect antigenicity of the E2 protein notably, since both the serine and proline mutants were readily detected by IFAT. (C) RT-PCR and sequencing of passage 3 of recombinant viruses confirmed that both the serine and the proline codons were stable.