Molecular determinants of Ebola virus virulence in mice

Abstract

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.

Figure 1. Molecular Differences among ZEBOV Mouse-Adapted Variants

(A) Comparison of ZEBOV variants. Pre–MA-ZEBOV differs from wild-type ZEBOV (WT-ZEBOV) by three amino acids in the GP (red triangles) and a silent mutation in the ORF of the VP40 gene (gray triangle). Serial passage of pre–MA-ZEBOV in progressively older mice yielded MA-ZEBOV [14], which contains coding changes in the NP, VP35, VP24, and polymerase (L) (all shown in red triangles). Two nucleotide changes localize to the NCRs at the 3′ end of the VP30 and the 5′ end of the VP24 gene (red triangles); the remaining three modifications in the NP, VP40, and L ORFs are silent (gray triangles). (B) Nucleotide and amino acid differences among WT-ZEBOV, pre–MA-ZEBOV, MA-ZEBOV, and MA-RG. The nucleotide changes in GP of pre–MA-ZEBOV, compared to WT-ZEBOV, as well as the changes acquired during adaptation of pre–MA-ZEBOV in mice are shown in red. −, no change.

Figure 2. Genetic Determinants of Virulence in Adult Mice

(A) Determination of MLD₅₀ values. Bars indicate the genotype of the viral gene: MA-ZEBOV (red), WT-ZEBOV (blue). The MLD₅₀ values of recombinant viruses were determined by i.p. inoculation of mice (three to six per group) with serial 10-fold dilutions of virus stock and then monitoring of survival rates. Experiments were carried out in duplicate. (B) Determination of the mean time to death and dose ranges causing morbidity/mortality. The mean time to death of mice inoculated with 10 FFU (approximately 1,000 MLD₅₀ for MA-ZEBOV) are indicated. Differences in the mean time to death for mice infected with various mutants, compared to that of mice infected with MA-ZEBOV, were considered significant when the p-value was <0.05. The dose range for morbidity/mortality was determined by inoculating groups of six to nine mice with the indicated amounts of viruses and monitoring the mice for weight loss and time to death. Survival numbers (dead/total) are color-coded to indicate the severity of infection in infected mice: no disease (black), illness without mortality (green), less than or equal to 50% mortality (purple), greater than 50% mortality (red). *Dead/total. **p < 0.05.

Figure 3. Growth Characteristics of Recombinant Viruses in Mice

Groups of 12 mice were inoculated i.p. with 5 FFU (approximately 500 LD₅₀ values for MA-ZEBOV) of representative viruses. On days 1, 2, 3, and 5 postinoculation, selected organs were removed from three infected animals per group. Virus titers in serum (A), spleen (B) and liver (C) were determined in Vero E6 cells by using a focus-forming assay [36].

Figure 4. Effect of Murine Type I IFNs on Recombinant Virus Replication in Mouse Macrophages

RAW 264.7 cells (mouse peritoneal macrophage-derived cell line) were infected with a multiplicity of infection of 0.05. Cells were untreated (A), treated with murine IFN-α/β (500 units/ml) 2 h postinfection (B), or treated with murine IFN-α/β (500 units/ml) 12 h prior to and again 2 h after virus adsorption (C). Supernatants were collected on days 0, 1, 2, 3, and 4 postinfection and titrated by use of a focus-forming unit assay in Vero E6 cells [36].