Mutagenesis of iBAC16 was performed as previously described (54). Briefly, the Kan/I-Sce I cassette was amplified from plasmid pEPKan-S by PCR. The purified PCR fragment was electroporated into iBAC16-containing GS1783 cells. Recombined clones were selected at 32°C on LB plates containing chloramphenicol (34 μg/ml) and kanamycin (50 μg/ml). Correct clones were then identified by PCR and confirmed by sequencing. Positive clones were induced at 42°C and plated on LB plates containing 1% l-arabinose and chloramphenicol (34 μg/ml) for secondary recombination. Clones from secondary recombination that were kanamycin sensitive were then picked and confirmed by PCR and sequencing. iBAC DNA was isolated using the Large Construct Kit (Qiagen) and used to transfect SLK cells with Lipofectamine 3000 (Thermo Fisher Scientific). SLK/iBAC cells were selected with hygromycin and induced with doxycycline (1 μg/ml) without or with sodium butyrate (1 mM) to generate recombinant virus. The sequences of primers are shown in Table 2.

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