Volume 23, Issue 6, Pages 1806-1816 (May 2018) Recently Identified Mutations in the Ebola Virus-Makona Genome Do Not Alter Pathogenicity in Animal Models  Andrea Marzi, Spencer Chadinah, Elaine Haddock, Friederike Feldmann, Nicolette Arndt, Cynthia Martellaro, Dana P. Scott, Patrick W. Hanley, Tolbert G. Nyenswah, Samba Sow, Moses Massaquoi, Heinz Feldmann  Cell Reports  Volume 23, Issue 6, Pages 1806-1816 (May 2018) DOI: 10.1016/j.celrep.2018.04.027 Copyright © 2018 Terms and Conditions

Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions

Figure 1 Phylogenetic Trees of EBOV Sequences Maximum likelihood phylogeny of 370 EBOV-Makona sequences (left: GP coding sequence only; right: full-length genome sequence tree rooted on EBOV-Kikwit and EBOV-Mayinga). Branches are colored according to the country of sample origin (legend in the upper left). Central bar indicates the predominant amino acid at position 82 in the EBOV GP gene. The numbers/letters in black represent the isolate identifiers. Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions

Figure 2 Infection Studies in IFNAR−/− Mice Groups of mice (4–7 weeks old; n = 12) were infected with 1,000 focus-forming units of EBOV-Mayinga, EBOV-Makona early 2014 (Guinea C05, C07, and C15), and EBOV-Makona later 2014 isolates (Mali 29; Liberia 304, 1225, 2596, 3697, and 4156). (A) Body weight curves (n = 8 per virus isolate) are shown. Error bars depict SEM. Black cross indicates time of death. Black asterisks represent ∗∗∗∗p < 0.0001 for EBOV-Mayinga versus each EBOV-Makona group. Blue asterisks represent significant differences between EBOV-Makona early and later 2014 groups at the level of ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05. (B) On day 4 after infection, animals (n = 4) per virus isolate were euthanized and samples were collected. Viral loads in the blood, liver, and spleen are depicted. Lines are representing the mean with SEM. Asterisks represent significant differences of EBOV-Mayinga versus each EBOV-Makona group at the level of ∗∗p < 0.01 and ∗p < 0.05. Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions

Figure 3 Influence of Virus Dose on IFNAR−/− Mice Infection Groups of mice (4 weeks; n = 6) were infected with different doses of EBOV-Mayinga 1976, an EBOV-Makona early 2014 (Guinea C15), or an EBOV-Makona later 2014 (Liberia 3697) isolate. (A–D) Survival and body weight curves are shown for infection with (A) 100,000 focus-forming units (FFUs), (B) 10,000 FFUs, (C) 1,000 FFUs, and (D) 100 FFUs. Error bars depict SEM. Black asterisks represent significant differences for EBOV-Mayinga versus each EBOV-Makona isolate; blue asterisks represent significant differences between EBOV-Makona early and later 2014 isolates. Significance levels are indicated as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions

Figure 4 Survival, Viremia, and Blood Chemistry Parameters in Infected Macaques Rhesus macaques were infected with 1,000 focus-forming units of EBOV-Mayinga 1976 (n = 5), two EBOV-Makona early 2014 isolates (Guinea C05, n = 3; Guinea C07, n = 3), and two EBOV-Makona later 2014 isolates (Mali 29, n = 3; Liberia 4156, n = 3). (A–D) Kaplan-Meier survival curves are shown for each individual isolate (n = 3) (A) and for grouped EBOV-Makona early 2014 (n = 6) and later 2014 (n = 6) isolates (C). Survival between EBOV-Makona later 2014 and EBOV-Mayinga was significantly different (p < 0.05, blue asterisk). Animals were examined and bled at the indicated time points post-infection. Viremia (titers) for individual (B) and grouped isolates (D) are shown. (E and F) The figure further shows serum concentration of interferon (IFN) α (E) and viral loads in selected tissues collected from macaques at the time of euthanasia (F). The asterisk represents a significant difference (∗p < 0.05). Error bars depict SEM. Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions

Figure 5 Coagulation Parameters, Interferon Response, and Tissue Viral Loads in Infected Macaques Rhesus macaques were infected with 1,000 focus-forming units of EBOV-Mayinga 1976 (n = 5), two EBOV-Makona early 2014 isolates (Guinea C05, n = 3; Guinea C07, n = 3), and two EBOV-Makona later 2014 isolates (Mali 29, n = 3; Liberia 4156, n = 3). Parameters are shown for EBOV-Mayinga 1976 (n = 5) and grouped EBOV-Makona early 2014 (C05 and C07; n = 6) and later 2014 (Mali 29 and Liberia 4156; n = 6). Animals were examined and bled at the indicated time points post-infection. (A–D) The figure displays coagulation parameters such as activated partial thromboplastin time (aPTT) (A), fibrinogen concentration (B), thrombin time (TT) (C), and plasma protein C (D). Significant differences in fibrinogen concentrations were found on day 4 between all EBOV-Makona isolates and EBOV-Mayinga (∗∗∗p < 0.001) and on day 8 between EBOV-Makona later 2014 isolates and EBOV-Mayinga (∗p < 0.05). (E–H) Further shown are platelet counts (E) and serum levels of blood urea nitrogen (BUN) (F), aspartate aminotransferase (AST) (G), and gamma-glutamyltransferase (GGT) (H) for the grouped isolates. The black asterisk represents significant difference in BUN on day 8 between EBOV-Makona isolates and EBOV-Mayinga (∗p < 0.05). Error bars depict SEM. Cell Reports 2018 23, 1806-1816DOI: (10.1016/j.celrep.2018.04.027) Copyright © 2018 Terms and Conditions