Plasmid 25CAT was linearized by XbaI and in vitro transcribed with T7 RNA polymerase to produce the DI RNA . The DI RNA was transfected into MHV-A59-infected DBT cells using DOTAP as described previously (Huang and Lai, 1999) . In brief,~80% con¯uent DBT cells were infected by MHV-A59 at an m.o.i. of 10. At 1 h p.i., the cells were transfected with 5 mg of in vitro transcribed DI RNA and incubated at 37°C for the desired lengths of time. To amplify the DI RNA, viruses (P0) were passaged twice in wt DBT cells to generate P1 and P2 viruses.