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

Murine macrophage cultures were isolated and maintained as described above. Cells were infected with EBOV GP/rVSV or MA-EBOV at an MOI of 0.1. When indicated, cells were incubated 15 minutes prior to and during infection with protein synthesis inhibitor cycloheximide (CHX) (Sigma) at a concentration of 10 μg/mL [91] . Total RNA was isolated using the mirVana miRNA isolation kit (Ambion-Life Technologies) at 2, 6, 14 or 24 hours following infection. RNA was quantified by Nanodrop (Thermo Scientific). Total RNA (300 ng) was reverse-transcribed to cDNA using random primers and M-MLV reverse transcriptase (Invitrogen) using manufacturer's specifications. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect transcript levels. SYBR Green based quantitative PCR reactions (Applied Bioscience) were performed using specific primers to VSV (M or L), EBOV (NP or L) [92] , proinflammatory cytokine/chemokines or ISGs and 2 uL of cDNA from each reaction. Primer sequences are available upon request. Expression levels were defined as the ratio between threshold cycle (Ct) values and the housekeeping gene, HPRT. The determined ratios were converted to log2 values. For the replication experiments, results are represented as fold change of log2 values calculated based on the 2 or 6 hours M-CSF value.