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, 90 (5), 2345-55

Determination and Therapeutic Exploitation of Ebola Virus Spontaneous Mutation Frequency

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Determination and Therapeutic Exploitation of Ebola Virus Spontaneous Mutation Frequency

Kendra J Alfson et al. J Virol.

Abstract

Ebola virus (EBOV) is an RNA virus that can cause hemorrhagic fever with high fatality rates, and there are no approved vaccines or therapies. Typically, RNA viruses have high spontaneous mutation rates, which permit rapid adaptation to selection pressures and have other important biological consequences. However, it is unknown if filoviruses exhibit high mutation frequencies. Ultradeep sequencing and a recombinant EBOV that carries the gene encoding green fluorescent protein were used to determine the spontaneous mutation frequency of EBOV. The effects of the guanosine analogue ribavirin during EBOV infections were also assessed. Ultradeep sequencing revealed that the mutation frequency for EBOV was high and similar to those of other RNA viruses. Interestingly, significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. In vitro, the presence of ribavirin increased the error rate, and the 50% inhibitory concentration (IC50) was 27 μM. In a mouse model of ribavirin therapy given pre-EBOV exposure, ribavirin treatment corresponded with a significant delay in time to death and up to 75% survival. In mouse and monkey models of therapy given post-EBOV exposure, ribavirin treatment also delayed the time to death and increased survival. These results demonstrate that EBOV has a spontaneous mutation frequency similar to those of other RNA viruses. These data also suggest a potential for therapeutic use of ribavirin for human EBOV infections.

Importance: Ebola virus (EBOV) causes a severe hemorrhagic disease with high case fatality rates; there are no approved vaccines or therapies. We determined the spontaneous mutation frequency of EBOV, which is relevant to understanding the potential for the virus to adapt. The frequency was similar to those of other RNA viruses. Significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. Ribavirin is a viral mutagen approved for treatment of several virus infections; it is also cheap and readily available. In cell culture, we showed that ribavirin was effective at reducing production of infectious EBOV. In mouse and monkey models of therapy given post-EBOV exposure, ribavirin treatment delayed the time to death and increased survival. These data provide a better understanding of EBOV spontaneous mutation and suggest that ribavirin may have great value in the context of human disease.

Figures

FIG 1
FIG 1
Effects of ribavirin on EBOV infectivity in vitro. EBOV was amplified in the presence of increasing concentrations of ribavirin, and plaque assays were used to determine the levels of infectious virus (measured as PFU per milliliter). Values shown represent the means for three biological replicates. Increasing concentrations of ribavirin resulted in decreased production of infectious virus. The IC50 was determined to be 27 μM. Ribavirin-induced cytotoxicity was not observed.
FIG 2
FIG 2
Weights of mice challenged with EBOV and prophylactically treated with ribavirin. Mice were infected with 1,000 PFU of mouse-adapted EBOV and treated with ribavirin 1 h prior to challenge and daily thereafter. Percent weight changes from baseline for each treatment group (80 mg/kg [a], 50 mg/kg, and 0 mg/kg [c]; n = 8 for each group) over the 21-day course of the study are shown. Means and standard errors of the means (SEM) are presented.
FIG 3
FIG 3
Survival of mice challenged with EBOV and prophylactically treated with ribavirin. Mice were infected with 1,000 PFU of mouse-adapted EBOV and treated with ribavirin 1 h prior to challenge and daily thereafter (n = 8 for each group). Survival over the course of the study is displayed. The experiment was performed one time, and two graphs are shown in order to compare each treatment group to the one untreated group. Ribavirin treatment correlated with increased survival and a significant delay to death compared to the results for the untreated group (P = 0.0001 for the 80-mg/kg group [a] and P < 0.0001 for the 50-mg/kg group). The median day to death for the untreated group was 6 days, while the median day to death for animals treated with 80 mg/kg was 12 days. Treatment with 50 mg/kg resulted in 75% survival, and treatment with 80 mg/kg resulted in 38% survival. While the 50-mg/kg group appeared to have slightly better outcomes than those of the 80-mg/kg group, there was no statistically significant difference in survival between the two groups (P = 0.185).
FIG 4
FIG 4
Weights of mice challenged with EBOV and treated with ribavirin postexposure. Mice were infected with 1,000 PFU of mouse-adapted EBOV and treated with different doses of ribavirin at 1 day postchallenge and daily thereafter (n = 8 for each group). (a) Four groups (n = 4 for each group) received the same treatment regimen but were left uninfected. Percent weight changes from baseline for each treatment group (30 mg/kg [e], 50 mg/kg [d], 100 mg/kg [c], 100-mg/kg loading dose with 50-mg/kg subsequent doses, and 0 mg/kg [f]) over the 21-day course of the study are shown. All groups exhibited a decline in weight over the first week. As the study progressed, many of the treated animals gradually regained weight. Means and SEM are presented for challenged animals. Means and data for each replicate are presented for unchallenged animals.
FIG 5
FIG 5
Survival of mice challenged with EBOV and treated with ribavirin postexposure. Mice were infected with 1,000 PFU of mouse-adapted EBOV and treated with different doses of ribavirin at 1 day postchallenge and daily thereafter (n = 8 for each group). Four groups (n = 4 for each group) received the same treatment regimen but were left uninfected. Survival over the course of the study is displayed for each treatment group compared to the untreated group. Postexposure ribavirin treatment correlated with increased survival and a significant delay in the median day to death (P = 0.004 for 30 mg/kg [a], P = 0.019 for 50 mg/kg, P = 0.0003 for 100 mg/kg [c], and P = 0.0003 for 100-mg/kg loading dose with 50-mg/kg subsequent doses [d]). The median day to death for the untreated group was 5 days, while the median day to death for treated animals was 7 days. The survival rate was 22%. There was no significant difference in survival or median day to death between the different treatment groups (P > 0.65).
FIG 6
FIG 6
Effect of daily postexposure ribavirin treatment on EBOV-challenged M. fascicularis macaques. Four M. fascicularis macaques were challenged i.m. with 100 PFU of EBOV. Three NHPs received twice-daily i.m. ribavirin treatment beginning at 1 day postinfection, and one NHP received saline as a control. During days 1 to 3, the treatment dose was 30 mg/kg, and on day 4, the dose was decreased to 15 mg/kg for the remainder of the study. (a) Treated animals exhibited lower serum titers than the untreated animal throughout the course of infection. (b) Treatment correlated with a delay in the median day to death (P = 0.083). The median day to death for the untreated NHP was 6 days, while the median day to death for treated NHPs was 8 days. (c) Activated partial thromboplastin times (aPTT) for blood collected on days 0, 3, 5, and 7 and at euthanasia. (d) Alkaline phosphatase (ALP) levels in blood collected on days 0, 3, 5, and 7 and at euthanasia. (e) Gamma glutamyltransferase (GGT) levels in blood collected on days 0, 3, 5, and 7 and at euthanasia. (f) Partial thromboplastin (PT) times for blood collected on days 0, 3, 5, and 7 and at euthanasia.

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