Interferon-? Inhibits Ebola Virus Infection

Rhein, Bethany A.; Powers, Linda S.; Rogers, Kai; Anantpadma, Manu; Singh, Brajesh K.; Sakurai, Yasuteru; Bair, Thomas; Miller-Hunt, Catherine; Sinn, Patrick; Davey, Robert A.; Monick, Martha M.; Maury, Wendy

Exosome IRF1 knockdown and EBOV GP/rVSV infections. BALB/c IFNAR -/peritoneal macrophages were isolated as described above and cultured 250,000 cells in a 96-well format in the presence of 50ng/mL M-CSF for 48 hours prior to addition of DsiRNA loaded exosomes. IRF1 or scrambled (Scr) siRNA loaded exosomes (2.5 μg) were added to macrophages in fresh media with fresh M-CSF with or without 20ng/mL IFNγ. Macrophages and exosomes were spinoculated by centrifugation at 700 x g at 4°C for 1 hour and then incubated at 37°C for 24 hours. Twenty-four hours later, all media, exosomes and cytokines were removed and macrophages were infected with fresh media containing EBOV GP/rVSV at an MOI of 0.1. Twenty-four hours following infection, cells were lysed as described above for RNA extraction. RNA isolation, cDNA synthesis and quantification of IRF1/VSV-L expression by qPCR were performed as described above. Expression levels were defined as the ratio between threshold cycle (Ct) values for IRF1 or VSV-L and the housekeeping gene, mouse HPRT, and is displayed as the log2 value of this ratio. In parallel experiments, other mice were administered IFNγ 2, 6, 12 or 48 hours following EBOV GP/rVSV infection by i.p. injection. In the VSV studies, post challenge administration of IFNγ was given only at 2 hours following infection. Mice were weighed and scored for sickness daily. Clinical sickness scoring was as follows: 0, no apparent illness; 1, slightly ruffled fur; 2, ruffled fur, active; 3, ruffled fur, inactive; 4, ruffled fur, inactive, hunched posture; 5, moribund or dead. Results represent scores from at least 10 mice per group. All mouse infection studies were concluded at 10 or 12 days following infection due to surviving mice regaining any lost weight and having no signs of clinical illness.