FIG 3.
Transfection-based inoculation of all-in-one bacmids leads to lethal PR8 virus replication in DBA mice. Five-week-old DBA/2J mice (n = 5 [A to D] or n = 15 [E to G]) were intranasally inoculated with 100 μl of inoculum containing 5 × 106 293T cells transfected 6 h earlier with bcmd-P8PR8 (H1N1PR8) or the corresponding 8-plasmid RG DNAs. Negative control mice received 5 × 106 mock-transfected 293T cells or cells transfected with either bcmd-P6PR8 without HA and NA cassettes or bcmd-DH10 empty DNA. Positive control mice were intranasally inoculated with wt PR8 virus (104 TCID50/100 μl). Mice were monitored for clinical signs, including body weight changes (A and E) and survival (B and F). At the time of death (C) or at 5 dpi (G), mice were necropsied or euthanized and lungs and brain were collected and analyzed for levels of virus in tissues by a TCID50 assay in MDCK cells. *, P < 0.05. A section of the collected tissues from panel C were subjected to IHC for detection of viral antigen (NP) (see Materials and Methods), which is indicated by the presence of a brown precipitate in panel D (representative staining is marked with arrows).