Linked Mutations in the Ebola Virus Polymerase Are Associated with Organ Specific Phenotypes.

Ebola virus (EBOV) causes a severe infection called Ebola virus disease (EVD). The pathogenesis of EBOV infection is complex, and outcome has been associated with a variety of immunological and cellular factors. Disease can result from several mechanisms, including direct organ and endothelial cell damage as a result of viral replication. During the2013 to 2016 Western Africa EBOV outbreak, several mutants emerged, with changes in the genes of nucleoprotein (NP), glycoprotein (GP), and the large (L) protein. Reverse genetic analysis has been used to investigate whether these mutations played any role in pathogenesis with mixed results depending on the experimental system used. Previous studies investigated the impact of three single nonsynonymous mutations (GP-A82V, NP-R111C, and L-D759G) on the fatality rate of mouse and ferret models and suggested that the L-D759G mutation decreased the virulence of EBOV. In this study, the effect of these three mutations was further evaluated by deep sequencing to determine viral population genetics and the host response in longitudinal samples of blood, liver, kidney, spleen, and lung tissues taken from the previous ferret model. The data indicated that the mutations were maintained in the different tissues, but the frequency of minor genomic mutations were different. In addition, compared to wild-type virus, the recombinant mutants had different within host effects, where the D759G (and accompanying Q986H) substitution in the L protein resulted in an upregulation of the immune response in the kidney, liver, spleen, and lungs. Together these studies provide insights into the biology of EBOV mutants both between and within hosts. IMPORTANCE Ebola virus infection can have dramatic effects on the human body which manifest in Ebola virus disease. The outcome of infection is either survival or death and in the former group with the potential of longer-term health consequences and persistent infection. Disease severity is undoubtedly associated with the host response, often with overt inflammatory responses correlated with poorer outcomes. The scale of the2013 to 2016 Western African Ebola virus outbreak revealed new aspects of viral biology. This included the emergence of mutants with potentially altered virulence. Biobanked tissue from ferret models of EBOV infected with different mutants that emerged in the Western Africa outbreak was used to investigate the effect of EBOV genomic variation in different tissues. Overall, the work provided insights into the population genetics of EBOV and showed that different organs in an animal model can respond differently to variants of EBOV.

Dose-Dependent Response to Infection with Ebola Virus in the Ferret Model and Evidence of Viral Evolution in the Eye.

Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.

Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.