Adult AG129 mice (129/Sv background deficient in alpha/beta interferon (IFN-α/β) and IFN-Ɣ receptors) were infected with recombinant Monkeypox virus (MPXV)expressing firefly luciferase by either intranasal (IN) or intraperitoneal (IP) routes. Experimental infections were conducted in a BSL-3 laboratory at the USGS National Wildlife Health Center, with a clade II MPXV that expresses firefly luciferase, as described in Osorio et. al, 2009 (MPXV/USA/luc). Twelve AG129 mice (8 males and 4 females) were infected intranasally with 105 plaque forming units (PFU) (5 ul, each nostril) of MPXV/USA/luc. Three control mice were inoculated in the same manner with an equal volume of PBS. Bioluminescent imaging was performed using an IVIS 200 series in vivo imager (Perkin Elmer, Oakland, CA). On days 19, 21, 23, and 25, post-infection, two to three mice were sacrificed to collect tissues. Twelve mice were also inoculated intraperitoneally with 105 PFU of the same virus diluted to 100 microliters of PBS. Three controls were injected intraperitoneally with the same volume of PBS. Two to three mice in this group were sacrificed on days 3, 5, 7, and 9 post-infection. Although tissues were collected, no further analysis was performed. Bioluminescent imaging was performed every other day from day 1 until day 29. Luminescence data was analyzed using Living Image software, version 3.2 (Perkin Elmer, Oakland, CA). Region of Interest (ROI) analysis was performed, using rectangular ROIs that covered the mouse body from nose to rump, excluding the tail unless significant luminescence was present on the tail. Luminescence within ROIs was quantified as total flux in photons per second (p/s).