Mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs after cardiac perfusion with 10 ml of PBS. Brains were separated into olfactory bulbs and the remainder of the brain, snap-frozen in liquid nitrogen (as well as livers and lungs) and RNA was extracted using TRIzol reagent (Invitrogen). DNase I treatment (DNAfree, Applied Biosystems/ Ambion) and reverse transcription with random hexamers (ImProm-II, Promega) were performed according to manufacturer's instructions. 0.5 ng of RNA was used in 384 well-format realtime PCRs in a Roche LightCycler 480 II using Applied Biosystem's SYBR Green PCR core reagents. PCR primers for murine ISG49/Ifit3, ISG54/Ifit2, ISG56/Ifit1 and 18S rRNA have been published previously [17] ; primers targeting murine Ifnb1 [59-CTTCTCCGTCATCTCCATAGGG-39 [39] , with the alternative reverse primer: 59-CACAGCCCTCTCCATCAACT-39], VSV N RNA [40] or EMCV 3D polymerase genomic region [41] were described previously. Primers for Ccl2, Il1b, Il6, Tnf, Il12b and Nos2 have been described previously [42, 43] . Average expression levels, relative to 18S rRNA and normalized by use of calibrator samples, were graphed with Prism 5.02 software. For analysis of different regions of the brain, brains without OB of perfused mice were separated into cortex, cerebellum, brain stem and remaining ''midbrain'', and tissue was submerged into RNAlater stabilizing reagent (Qiagen) overnight and frozen. RNA was then extracted via TRIzol and further processed and assayed by realtime RT-PCR as described above. For microarray analysis, TRIzol-extracted and DNase I-treated RNA was additionally purified using spin columns (RNeasy Mini kit, Qiagen) before subjection to mRNA expression microarray analysis via Illumina Mouse Ref-8 V2 beadchip and GenomeStudio software V2010.2 (Illumina, Inc.); RNA hybridization to chips was performed by the Lerner Research Institute Genomics Core at the Cleveland Clinic. Microarray raw data were deposited in the NCBI Gene Expression Omnibus (GEO), accession number GSE33678.