FIG. 5.

Effects of amino acid substitutions at Q6327 on IBV replication. (A) Growth properties of the Q6327H mutant virus. Vero cells were infected with wild-type and mutant viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, 24, and 36 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID₅₀ of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (B) Detection of genomic and subgenomic RNA synthesis in cells electroporated with full-length Q6327P mutant transcripts. Total RNA was prepared from Vero cells electroporated with in vitro-synthesized full-length transcripts at 48 h postelectroporation. Regions corresponding to nucleotides 14931 to 15600 of the positive (+)- and negative (−)-sense IBV genomic RNA, and the 5′-terminal 415 and 1010 nucleotides of subgenomic RNA 4 and 3, respectively, were amplified by RT-PCR and analyzed on 1.2% agarose gel. Lanes 1 and 9 show DNA markers. The sizes of the molecular weight markers are indicated on the left.