Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance

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Science  21 Nov 2014:
Vol. 346, Issue 6212, pp. 987-991
DOI: 10.1126/science.1259595


Existing mouse models of lethal Ebola virus infection do not reproduce hallmark symptoms of Ebola hemorrhagic fever, neither delayed blood coagulation and disseminated intravascular coagulation nor death from shock, thus restricting pathogenesis studies to nonhuman primates. Here we show that mice from the Collaborative Cross panel of recombinant inbred mice exhibit distinct disease phenotypes after mouse-adapted Ebola virus infection. Phenotypes range from complete resistance to lethal disease to severe hemorrhagic fever characterized by prolonged coagulation times and 100% mortality. Inflammatory signaling was associated with vascular permeability and endothelial activation, and resistance to lethal infection arose by induction of lymphocyte differentiation and cellular adhesion, probably mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever.

Variety of Ebola symptoms in mice

Apart from monkeys, there are no animal models available that show the same symptoms of Ebola virus infection as those of humans. Rasmussen et al. tested the effects of Ebola virus in mice with defined genetic backgrounds in a series of pains-taking experiments performed under stringent biosafety conditions. Resistance and susceptibility to Ebola virus was associated with distinct genetic profiles in inflammation, blood coagulation, and vascular function. This panel of mice could prove valuable for preliminary screens of candidate therapeutics and vaccines.

Science, this issue p. 987

A mouse-adapted strain of Ebola virus (MA-EBOV) does not cause hemorrhagic syndrome despite causing lethal disease in laboratory mice, and it cannot be used effectively to study Ebola hemorrhagic fever (EHF) pathogenesis, because the dissimilarity to human disease limits the ability to identify key correlates of viral pathogenesis or accurately assess the effect of vaccines or therapeutics. Pathogenesis studies of EHF have thus been restricted to macaques (14), guinea pigs (5, 6), and Syrian hamsters (7). Although these models accurately recapitulate most of the disease features of EHF, practical and ethical concerns limit their use, including nonreproducible genetic backgrounds, cost, animal availability, and reagent availability. Epidemiologic studies of EBOV infection have identified a range of pathogenic phenotypes, which are not linked to specific mutations in the viral genome (8, 9). This suggests that the host response may determine disease severity after EBOV infection.

We tested the role of host genetics in Ebola virus disease (EVD) using the Collaborative Cross (CC) resource, a genetically diverse panel of recombinant inbred (CC-RI) mice obtained through a systematic cross of eight inbred founder mouse strains, five of which are classic laboratory strains (C57BL/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/H1LtJ) and three of which are wild-derived inbred strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ) (10). The founders represent 90% of the common genetic variation across the three major Mus musculus subspecies (M. m. musculus, M. m. domesticus, and M. m. castaneus) (11). Different strains can be crossed with one another to generate CC-RI intercrossed (CC-RIX) F1 progeny. We recently observed a spectrum of pathogenic phenotypes in CC mice and identified genetic loci associated with influenza severity and disease outcome (12, 13). Thus we tested whether a similar range of phenotypes would emerge after infecting CC-RIX animals with MA-EBOV.

To determine a phenotypic baseline, we challenged the eight CC founders intraperitoneally with MA-EBOV or the Mayinga strain of wild-type EBOV (WT-EBOV). MA-EBOV differs from the published WT-EBOV sequence by only 13 nucleotide changes, three of which are silent (14). MA-EBOV is pathogenic in guinea pigs and macaques (1) and causes lethal EHF in Syrian hamsters (7). Despite observing 25 to 100% mortality after MA-EBOV challenge at multiple doses (fig. S1), we found no evidence of hemorrhagic disease or susceptibility to lethal disease after infection with WT-EBOV. We assessed the pathogenic phenotype produced by intraperitoneal infection with 100 focus-forming units (FFUs) of MA-EBOV in 47 available CC-RIX lines (Table 1). We observed disease phenotypes ranging from complete resistance to lethal disease to severe EHF-associated pathology before death, as well as lines that showed lethal infection without symptoms of EHF but sometimes with hepatic discoloration.

Table 1

Distribution of phenotypes across CC-RIX lines. Boldface type indicates CC-RIX crosses used in this study.

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We performed detailed studies on two representative lines, 13140x3015 (susceptible to lethal EHF) and 15156x1566 (resistant to lethal disease). Mice from both lines lost approximately 15% of their body weight over the first 5 days post-infection (p.i.) (Fig. 1A). However, susceptible mice succumbed to lethal infection on days 5 to 6 p.i., whereas resistant mice survived and fully recovered their body weight by day 14 (Fig. 1B). At day 5 p.i., susceptible mice presented pathological findings consistent with EHF, including prolonged blood coagulation, internal hemorrhage, coffee-colored blood, splenomegaly, and hepatic discoloration and softened texture (Fig. 1C). The resistant mice, however, had no evident gross pathology at the time of maximum body weight loss and no alteration in the appearance of the liver (Fig. 1D). Neither susceptible nor resistant mice developed observable clinical disease after challenge with WT-EBOV. We detected extremely low titers of virus at day 3 in the liver and spleen of animals after WT-EBOV infection, and these were 100 to 1000 times lower than organ titers detected in mice infected with MA-EBOV (fig. S2). We did not detect virus at day 5 in any organ or any mouse, indicating that WT-EBOV is not able to productively replicate in these mouse strains.

Fig. 1

Distinct morbidity and mortality after MA-EBOV infection in CC-RIX mouse lines. (A) Percent of starting body weight over the course of infection in susceptible (red squares) and resistant (blue circles) mice. Data shown are mean ± SEM from five mice per CC-RIX line. (B) Kaplan-Meier survival curve for susceptible (red) and resistant (blue) mice. Five mice were used for each CC-RIX line. (C to F) Gross appearance of liver at necropsy in uninfected susceptible (C) and resistant (E) mice and on day 5 p.i. in susceptible (D) and resistant (F) mice.

In liver and spleen from both mouse lines, equivalent levels of viral RNA were observed (Fig. 2, A and B). However, we observed 1 to 2 logs higher levels of infectious virus in susceptible liver and spleen than in resistant liver and spleen after virus titration by focus-forming assay when infectious virion production became detectable on day 3 (Fig. 2, C and D), suggesting that resistance may be associated with a defect in virion assembly, secretion, or other posttranscriptional processes. We confirmed this finding by staining liver sections from susceptible and resistant mice on day 5 p.i. for VP40, the viral matrix protein. We observed substantially less VP40 staining in resistant liver (Fig. 2, E and F) than in susceptible liver (Fig. 2, G and H, and fig. S3). Sequence analysis showed no nucleotide changes between virus genomes in either line, indicating that these effects cannot be readily attributed to the selection of quasispecies with different viral fitness (table S1). Despite significant differences in infectious virus titers between the two mouse lines, we observed similar levels of inflammation and apoptosis in the spleen and liver, although the two lines displayed distinct histopathology (figs. S4 to S6). Despite similar organ tropism, virus infection occurred in different hepatic cell types in the two mouse lines. Susceptible mice had viral antigen in essentially every hepatocyte (Fig. 2F and table S2); whereas in resistant mice, viral antigen was restricted to cells that lack typical hepatocyte morphology, most likely endothelial cells and Kupffer cells (Fig. 2G), consistent with low-pathogenicity Reston virus infection (15). Possibly in resistant mice, infected hepatic endothelial cell and macrophage responses limit virus production and control systemic inflammation and coagulopathy. Widespread hepatic infection in susceptible mice may explain how these animals both produce increased amounts of infectious virus and induce dysregulated coagulation pathways.

Fig. 2

MA-EBOV replication in CC-RIX mouse lines. (A and B) Quantitative real-time polymerase chain reaction showing the expression of MA-EBOV genomes relative to mouse 18S ribosomal RNA in spleen (A) and liver (B). Data shown are mean ± SEM for three mice per time point per RIX line. (C and D) Titration of infectious MA-EBOV in organ homogenates from spleen (C) and liver (D) quantified as FFUs per milliliter. No infectious virus was detected before day 3 p.i. Data shown are mean ± SEM from two experiments using two or three mice per time point per CC-RIX line. (E to H) Immunohistochemical staining for VP40 in resistant liver [(E) and (F)] and susceptible liver [(G) and (H)]. The arrows indicate representative hepatocyte morphology (t test, *P < 0.05).

We quantified the extent of coagulopathy by measuring blood clotting times. On days 5 to 6 p.i., susceptible mice showed significantly prolonged thrombin time (TT), prothrombin time (PTT), and activated partial thromboplastin time (aPTT) compared to resistant and C57BL/6J mice (Fig. 3, A to C). An initial spike in serum fibrinogen levels in susceptible mice on day 3 p.i. was followed by a precipitous drop (Fig. 3D) before death. This increase may be due to compensatory fibrinogen production in response to hepatic cell death and consequent clotting factor depletion, which is consistent with observations in other EHF models in which severe hemorrhage and coagulopathy typically peak within 48 hours preceding death (3, 7).

Fig. 3 Quantification of coagulopathy and hemorrhage in CC-RIX mouse lines.

(A to C) Coagulation times in seconds for thrombin (A), prothrombin (B), and activated partial thromboplastin (C) over the course of MA-EBOV infection. (D) Serum fibrinogen levels in CC-RIX mice over the course of MA-EBOV infection. All data shown are the mean ± SEM for two experiments including two to five animals per time point. (ANOVA with Tukey’s HSD post-hoc test; * P < 0.05, ** P < 0.05, *** P < 0.0000001).

We investigated transcriptional host responses linked to disease outcome in the CC-RIX lines. Significant differentially expressed genes relative to time-matched mock-infected samples (false discovery rate–adjusted P value <0.05; fold change >1.5) in both spleen and liver were 10 to 100 times higher in number in susceptible mice than in resistant mice (Fig. 4, A and B, and supplementary data files S2 and S3). These data suggest that EHF is characterized by earlier induction of a larger-magnitude transcriptional response. In susceptible mice relative to resistant mice, genes associated with EBOV infection were differentially induced. Early in infection in the spleens of susceptible mice at day 1 p.i., we observed enrichment of p38 mitogen-activated protein kinase and extracellular signal–regulated kinase signaling, processes that stimulate productive EBOV infection (16, 17). Additionally, we observed increased nuclear factor κB expression and the induction of proinflammatory processes, which may reflect early targets of infection in the secondary lymphoid organs. By day 3 p.i. in both liver and spleen, inflammatory pathways became increasingly enriched in susceptible mice, as did pathways associated with cell death, including those associated with cytotoxicity and apoptosis in macrophages and endothelial cells. Both resistant and susceptible lines induced multiple immune pathways in the spleen. By day 5, although differential gene expression peaked in both lines, the gene sets involved were distinct and probably reflected different courses of disease.

Fig. 4

Distinct host responses associated with disease phenotype. (A and B) Number of differentially expressed genes either up-regulated (positive y axis) or down-regulated (negative y axis) relative to time-matched mock-infected samples in spleen (A) and liver (B).

We identified differentially expressed genes unique to susceptible mice in the liver and observed enrichment in genes related to vascular integrity at days 3 and 5, including the endothelial tyrosine kinases Tie1 and Tek (Tie2). Tie1 and Tek expression was depressed as compared with levels in mock-infected animals at day 5, concurrent with the onset of coagulopathy. We used Ingenuity Pathway Analysis software to generate networks predicting molecular activity (18) and predicted the activation of processes associated with vascular differentiation and endothelial activation, interleukin-6–mediated inflammation and bleeding, and the inhibition of pathways associated with vascular integrity and inflammatory regulation in susceptible livers (fig. S7). TIE1 and TEK signaling promotes the activation of coagulation factors, such as thrombin (F2), tissue factor (F3), and protease-activated receptors 1, 3, and 4 (PAR1/F2R, PAR3/F2RL2, and PAR4/F2RL3) (19), which have been mechanistically implicated in coagulopathies mediated by EBOV and other viruses (4, 20) and are differentially regulated in these mice (fig. S8). Tie1 and Tek expression was consistently elevated in resistant mouse spleens, implying that endothelial signaling regulation and vascular leakage contribute to disease resistance in susceptible mice.

In livers from resistant mice at day 5, gene expression associated with vascular density and angiogenesis increased, suggesting that this line effectively controls vascular leakage, potentially through repair or structural maintenance of blood vessels. It seems likely that restriction of MA-EBOV infection to endothelial and Kupffer cells in resistant mice prevents the induction of hepatocyte-specific molecules that enhance systemic inflammation, thrombocytopenia, and coagulopathy.

We investigated the genomes and found that the Tie1 alleles across the eight CC founders are from all three M. musculus subspecies and are highly divergent from one another (21), which prevented us from identifying significant relationships between Tie1 alleles and phenotypes. In contrast, Tek alleles in the CC-RIX lines are derived from only two subspecies, M. m. domesticus and M. m. musculus, and are very different from one another. Distinct Tek alleles were previously associated with inflammatory coagulopathies and vascular dysfunction (2226). In our preliminary analysis, we identified statistically significant relationships between subspecific Tek alleles and the initial onset of weight loss [analysis of variance (ANOVA), F2,31 = 5.581, P = 0.0085], average day of death (ANOVA, F2,34 = 10.519, P = 0.00028), and mortality (ANOVA, F2,37 = 8.5553, P = 0.0008) (fig. S9).

We reproduced EHF in a mouse model that will allow the linkage of specific genetic polymorphisms to tropism, infectious virus production, cell type–specific responses, and phenotypic outcome. The CC model provides a platform to map susceptibility alleles in the context of EHF pathogenesis and rapidly apply these findings to the development of candidate therapeutics and vaccines. Ongoing screening activities in CC-RIX mice will identify additional genetic loci that contribute to hemorrhagic disease, lethality, or resistance to severe disease.

The frequency of different pathological manifestations across the 47 CC-RIX lines screened so far is similar in variety and proportion to the spectrum of clinical disease observed in patients with EVD in the 2014 West Africa outbreak, with hemorrhagic symptoms appearing in 30 to 50% of patients (27, 28). Although we cannot rule out the possibility that human survivors have preexisting immunity to EBOV or a related virus, our data suggest that genetic factors play a significant role in determining disease outcome in naïve individuals without prior exposure or immunologic priming.

Although we have not yet screened CC-RIX mice for susceptibility to other ebolavirus species, we anticipate that we would observe a similar distribution of pathogenic phenotypes after infection with viruses that are capable of replicating in mice. The current 2014 West Africa outbreak is caused by the same species of ebolavirus as the MA-EBOV used in this screen. There are also similarities in the spectrum of disease observed in CC-RIX mice infected with MA-EBOV and in clinical cases in the current outbreak. The model described in this paper can be implemented promptly to identify genetic markers, conduct meticulous pathogenesis studies, and evaluate therapeutic strategies that have broad-spectrum antiviral activity against all Zaire ebolaviruses, including the virus responsible for the current West Africa outbreak.

Supplementary Materials

Materials and Methods

Figs. S1 to S9

Tables S1 to S3

Data Files S1 to S7

References and Notes

  1. Supplementary text.
  2. Acknowledgments: This study was supported in part by awards U54 AI081680, U19 AI109761, and U19 AI100625 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health; P51 OD010425 from the Office of the Director, National Institutes of Health; and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Microarray data have been deposited with the Gene Expression Omnibus ( (accession number GSE57214), and raw data can be obtained at Mice were developed by the Collaborative Cross Consortium, an international consortium of researchers who designed, planned, and implemented the Collaborative Cross project. Initial animals were provided by the Jackson Laboratory, and CC lines were developed independently at the Oak Ridge National Laboratories in Tennessee, USA; Tel Aviv University, Israel; and Geniad, Ltd., Australia. CC lines are currently produced and distributed at the University of North Carolina, Chapel Hill. A.L.R. designed the study, performed functional analysis of microarray data, and wrote the manuscript; A.O. performed infections, veterinary examinations, and necropsies and assessed phenotype, collected and processed samples, and titrated virus from organs by focus-forming assay; M.T.F., M.T.H., F.P.-M.V., and R.S.B. established systems for designing and breeding CC-RIX mouse populations and utilizing them for virus pathogenesis studies and contributed to strain selection and data analysis; R.G. performed microarray data normalization, batch correction, and differential expression analysis; S.M.K. and J.M.W. performed target preparation and hybridization of microarrays; R.L. coordinated veterinary care for experimental animals; D.P.S. performed histopathological staining and analyzed the histopathology data; F.F., D.S., and E.H. assisted with mouse procedures in high biocontainment; M.J.T. and R.G. performed sequencing and subsequent analysis of viral RNA; A.F. performed functional analysis of microarray data; P.S. quantified viral RNA by quantitative polymerase chain reaction; M.J.K. edited the manuscript; and H.F. and M.G.K. contributed significantly to study design, provided space and infrastructure for the experiments and analysis, assisted in data analysis, and edited the manuscript.
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