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The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies.

Ebola virus (EBOV) is a filovirus that can cause Ebola virus disease (EVD). No approved vaccines or therapies exist for filovirus infections, despite an urgent need. The development and testing of effective countermeasures against EBOV requires use of animal models and a thorough understanding of how the model aligns with EVD in humans. The majority of published studies report outcomes of parenteral exposures for emulating needle stick transmission. However, based on data from EVD outbreaks, close contact exposures to infected bodily fluid seems to be one of the primary routes of EBOV transmission. Thus, further work is needed to develop models that represent mucosal exposure. To characterize the outcome of mucosal exposure to EBOV, cynomolgus macaques were exposed to EBOV via intranasal (IN) route using the LMA ® mucosal atomization device (LMA ® MAD). For comparison, four non-human primates (NHPs) were exposed to EBOV via intramuscular (IM) route. This IN exposure model was uniformly lethal and correlated with a statistically significant delay in time to death when compared to exposure via the IM route. This more closely reflects the timeframes observed in human infections. An IN model of exposure offers an attractive alternative to other models as it can offer insight into the consequences of exposure via a mucosal surface and allows for screening countermeasures via a different exposure route., Competing Interests: The authors declare no conflict of interest. The funding agency had no role in the design, collection, analyses, or interpretation of these data; in the writing of the manuscript, and in the decision to publish the results.

Sudan virus (SUDV), like the closely related Ebola virus (EBOV), is a filovirus that causes severe hemorrhagic disease. They both contain an RNA editing site in the glycoprotein gene that controls expression of soluble and full-length protein. We tested the consequences of cell culture passage on the genome sequence at the SUDV editing site locus and determined whether this affected virulence. Passage resulted in expansion of the SUDV editing site, similar to that observed with EBOV. We compared viruses possessing either the wild-type or expanded editing site, using a nonhuman primate model of disease. Despite differences in virus serum titer at one time point, there were no significant differences in time to death or any other measured parameter. These data imply that changes at this locus were not important for SUDV lethality., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: This study addresses the role of Ebola virus (EBOV) specific infectivity in virulence. Filoviruses are highly lethal, enveloped, single-stranded negative-sense RNA viruses that can cause hemorrhagic fever. No approved vaccines or therapies exist for filovirus infections, and infectious virus must be handled in maximum containment. Efficacy testing of countermeasures, in addition to investigations of pathogenicity and immune response, often requires a well-characterized animal model. For EBOV, an obstacle in performing accurate disease modeling is a poor understanding of what constitutes an infectious dose in animal models. One well-recognized consequence of viral passage in cell culture is a change in specific infectivity, often measured as a particle-to-PFU ratio. Here, we report that serial passages of EBOV in cell culture resulted in a decrease in particle-to-PFU ratio. Notably, this correlated with decreased potency in a lethal cynomolgus macaque (Macaca fascicularis) model of infection; animals were infected with the same viral dose as determined by plaque assay, but animals that received more virus particles exhibited increased disease. This suggests that some particles are unable to form a plaque in a cell culture assay but are able to result in lethal disease in vivo. These results have a significant impact on how future studies are designed to model EBOV disease and test countermeasures., Importance: Ebola virus (EBOV) can cause severe hemorrhagic disease with a high case-fatality rate, and there are no approved vaccines or therapies. Specific infectivity can be considered the total number of viral particles per PFU, and its impact on disease is poorly understood. In stocks of most mammalian viruses, there are particles that are unable to complete an infectious cycle or unable to cause cell pathology in cultured cells. We asked if these particles cause disease in nonhuman primates by infecting monkeys with equal infectious doses of genetically identical stocks possessing either high or low specific infectivities. Interestingly, some particles that did not yield plaques in cell culture assays were able to result in lethal disease in vivo. Furthermore, the number of PFU needed to induce lethal disease in animals was very low. Our results have a significant impact on how future studies are designed to model EBOV disease and test countermeasures.