IFN⍺R1-/- and WT C57BL/6N mice were housed in ventilated racks with free access to food and water and maintained on a 12 h light/dark cycle. WT C57BL/6N mice were purchased from Jackson Laboratories and IFN⍺R1-/- were used from an established breeding colony at the VGTI/OHSU. Animals were vaccinated with 100 μl AdV-MAYV (1x106 to 1x108 plaque forming units (PFU)) diluted in PBS injected into the posterior thigh muscle with or without a boost at 14 days post-prime using the same viral dose. At times listed in the figure legends, blood was collected from the facial vein and allowed to clot before centrifuging for 10 minutes at 9,391 x g in a microcentrifuge in order to collect serum. Vaccinated mice were challenged with virus at either 28- or 84-days post-vaccination (dpv). A few groups of animals received passive transfer of 200 μl of pooled serum from five AdV-MAYV or mock vaccinated mice by intraperitoneal injection at 24 h before infection. Mice were challenged with 104 PFU of MAYVBeAr, UNVMAC 150, or 103 PFU CHIKVSL15649, via a 20 μl injection into the right posterior footpad. Footpad swelling measurements were performed with digital calipers and health and weight were monitored daily following challenge. Serum was collected at 2 days post-infection (dpi) to measure viremia by plaque assay. These challenge studies were terminated at either 4 or 7 dpi at which time ankle, calf muscle, quadricep muscle, spleen, and serum were collected for use in plaque titration assays and qRT-PCR as previously described [39]. Tissue samples were collected into tubes with 0.5 ml of PBS and 2 mm beads (Propper Manufacturing Co., Inc.) for homogenization by bead beating using a Precellys 24 homogenizer (3 times x 45 seconds; Bertin Technologies). Cellular debris were pelleted and clarified lysate was used for infectious virus titration and cytokine multiplex assays. An additional 100 μl aliquot of the tissue homogenate was added to Trizol for qRT-PCR analysis. Histological analysis was performed on groups of five-week-old female WT C57BL/6N mice that received passive transfer of naïve or immune serum from vaccinated animals. One day after transfer, animals were challenged with 104 PFU MAYVBeAr in the right hind limb footpad. A second group of naïve five-week-old female WT C57BL/6N were mock inoculated with PBS.

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