Generation of Ebolavirus (EBOV)/Makona-mouse-adapted (MA) by serial passage in suckling BALB/c mice. (A) The EBOV/Makona-preMA genome consisted of six mutations associated with EBOV/Mayinga-MA that were inserted into the coding regions of NP, GP, VP24, and L genes (purple arrows). An additional EBOV/Mayinga-MA mutation in the non-coding region (NCR) of VP24 was inserted (gray arrow). Two EBOV/Mayinga-MA mutations already existing in the EBOV/Makona genome are denoted (blue star). Two additional mutations in NP and VP24 were identified in EBOV/Makona-MA after serial passaging (red triangles). (B) Serial passage of EBOV/Makona-preMA in progressively older suckling BALB/c mice resulted in the production of EBOV/Makona-MA. For each passage, livers were collected, pooled, and used to infect a new set of naive mice. Corresponding viral loads were measured by RT-qPCR for EBOV L (mean Ct values shown), *no livers were collected from P9 mice as all mice succumbed to infection.