FIG 7.

IFN-γ-treated MH-S murine alveolar macrophages are resistant to EHV-1 infection. (A) EHV-1 strains KyA and RacL11 replicate in MH-S murine alveolar macrophages (CRL-2019; American Type Culture Collection). Virus titers were determined at 0, 24, and 48 hpi. MH-S cells (1 × 10⁶) were infected with KyA or RacL11 (multiplicity of infection [MOI], 1). Intracellular virus was released by three freeze-thaw cycles at 0, 24, or 48 hpi, and titers were determined by plaque assays, which were performed as described previously (50). Each sample was assayed in triplicate. Error bars indicate standard deviations. (B) MH-S cells were infected with KyA or RacL11 at an MOI of 5, harvested at 6 hpi, and used for Western blot analyses with the anti-IEP polyclonal antibody OC33 (77) and an anti-EICP0 polyclonal antibody (28). The sizes of molecular mass standards (Bio-Rad Laboratories, Hercules, CA) are given on the left. (C and D) IFN-γ reduced EHV-1 gene expression. (C) MH-S cells were treated with 0.002, 0.02, 0.2, or 2 ng/ml of murine IFN-γ (485-MI; Cell Sciences, Canton, MA) and were infected with EHV-1 RacL11 (MOI, 5) at 24 h posttreatment. The infected cells were harvested at 24 hpi and were used for Western blot analyses with the anti-IEP polyclonal antibody OC33 (77), anti-ETIF (78), and anti-actin (SC-1615-R; Santa Cruz Biotechnology, Santa Cruz, CA). The sizes of molecular mass standards (in kilodaltons) (Bio-Rad Laboratories, Hercules, CA) are given on the left. (D) MH-S cells were treated with 2 or 20 ng/ml of murine IFN-γ and were infected with EHV-1 RacL11 (MOI, 5) at 24 h posttreatment. The infected cells were harvested at 6 or 24 hpi for Western blot analyses using antibodies to the early proteins UL5 (79) and IR4 (80). (E to G) Virus titers were determined by plaque assays on NBL6 cells. (E) MH-S cells (1 × 10⁶) were treated with 0.02, 0.2, 2, or 20 ng/ml of IFN-γ, infected with EHV-1 RacL11 (MOI, 1) at 24 h posttreatment, and washed four times with growth medium without serum. Intracellular virus was released by three freeze-thaw cycles at 24 hpi. (F and G) MH-S cells (1 × 10⁶) were treated with 20 ng/ml of IFN-γ, infected with wild-type EHV-1 Ab4 (MOI, 1) (F) or EHV-1 KyA (MOI, 1) (G) at 24 h posttreatment, and washed four times with growth medium without serum. Each sample was assayed in triplicate. Error bars indicate standard deviations.