In this study, we have identified molecular determinants of ZEBOV virulence in mice by using reverse genetics. We found VP24 and NP to be the primary determinants for adaptation of WT-ZEBOV in mice and found a correlation between virulence and the ability of the virus to evade the type I IFN-induced antiviral response. The ability to overcome the IFN-induced antiviral response is, therefore, a critical event in the pathogenesis of ZEBOV infection in mice and possibly in humans. Moreover, mutations in other viral proteins and NCRs also contribute to the virulent phenotype, indicating that virulence is a multifactor trait.
Recent in vitro studies suggest that VP24 functions as a type I IFN antagonist [7
], but the significance of this finding had not been addressed in vivo until now. Here, we have demonstrated that single amino acid modifications in VP24 and NP of MA-ZEBOV are critical for virus evasion of IFN-induced antiviral responses. NP has not previously been considered as a potential IFN antagonist. However, given that it interacts with VP24 in the formation of nucleocapsids [27
], it is not unreasonable to imagine that it may act as an enhancer and/or stabilizer of VP24 functions. Nevertheless, since the NP mutation acquired during mouse adaptation of WT-ZEBOV alone allowed the recombinant virus to replicate in IFN-treated cells (see C), NP likely plays a more direct role in the evasion of the IFN-induced antiviral response. Interestingly, WT-NP/VP24MA
grew more efficiently than MA-RG in IFN-stimulated cells, in contrast to our in vivo results. For this reason, it seems likely that the mutations in VP24 and NP are critical for resistance to IFN-induced antiviral responses, while the remaining mutations acquired during adaptation of WT-ZEBOV facilitate efficient virus replication and/or spread in mice despite their attenuated phenotype in cell culture systems.
VP35 and GP have previously been linked to EBOV pathogenicity [4
]. VP35 is an IFN antagonist that interferes with type I IFN synthesis by inhibiting IRF-3 (interferon regulatory factor 3) activation, a necessary step for the transcription of IFN genes [9
]. Although the mutation found in the mouse-adapted VP35 protein was not responsible for the enhanced virulence of MA-ZEBOV (B), this does not necessarily indicate that VP35-mediated regulation of IFN levels does not play a part in the pathogenicity of EBOV. This activity may still be important for EBOV to achieve high virulence. Likewise, GP is cytotoxic and is, therefore, thought to contribute to viral pathogenicity [5
]. However, a recent report suggested that this cytotoxicity originated from overexpression of GP in cells [11
]. As with VP35, the lack of an adaptive mutation in the GP of MA-ZEBOV does not necessarily diminish the role of GP in viral pathogenicity.
Interestingly, the adaptation of ZEBOV in guinea pigs also resulted in amino acid changes in NP, VP24, and L and in a nucleotide substitution in the VP30 NCR [8
]. Although these mutations differed in their amino acid positions from those in MA-ZEBOV, it seems likely that they serve a similar role in adaptation and also function by counteracting innate antiviral responses [31
]. As with mice, additional mutations (e.g., in L and the VP30 NCR) likely contribute to virulence by affecting viral transcription/replication.
The mouse model using MA-ZEBOV does not exactly mirror all aspects of human Ebola hemorrhagic fever [15
]. Thus, determinants for virulence of ZEBOV may differ between mouse and primate models. However, since ZEBOV is naturally lethal to primates, but not to mice, this model provided an opportunity to decipher the roles of viral proteins in expression of high virulence in a host (i.e., mice). Our studies showed that ZEBOV VP24 and NP are inadequate for the expression of high virulence in mice, but upon mutation, optimally expressed this property. Thus, it is possible that these viral proteins play an important role in expression of high virulence in primates. Likewise, the lack of VP35 mutations correlating with pathogenicity in mice is interesting, indicating that ZEBOV VP35 optimally functions in both primates and mice without additional mutation in an IFN pathway. Since MA-ZEBOV is attenuated in NHPs [18
], one or more genes into which mutations were introduced during mouse adaptation of ZEBOV likely play a role in virulence in primates. Therefore, it will be interesting to examine the virulence of selected recombinant mouse-adapted variants in NHPs. Such studies will provide us with valuable information for understanding ZEBOV pathogenesis in humans.
Of note, we observed resistance in mice to infection with high infectious doses of MA-ZEBOV (B). A similar finding has been observed in a mouse model for rabies virus [33
]. The most likely explanation for this finding is that inoculation with high doses of virus causes a rapid stimulation of the innate immune response before virus replication or spread can occur. This topic presents an attractive research subject that may lead to control measures for EBOV infections through immunologic modulation of host responses to viral infection.
In conclusion, the combination of reverse genetics technology [34
] and a small-animal model has allowed us to gain valuable insights into EBOV pathogenesis. Understanding the molecular basis of mouse adaptation of ZEBOV will likely lead to the identification of viral genetic determinants of EBOV virulence and to the elucidation of the roles of specific viral proteins in the pathogenic process. A more detailed molecular understanding of virulence and the host responses will also be crucial to improving our ability to control EBOV infections in the future.