Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses.

Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.

Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Smallpox lesion characterization in placebo-treated and tecovirimat-treated macaques using traditional and novel methods.

Smallpox was the most rampant infectious disease killer of the 20th century, yet much remains unknown about the pathogenesis of the variola virus. Using archived tissue from a study conducted at the Centers for Disease Control and Prevention we characterized pathology in 18 cynomolgus macaques intravenously infected with the Harper strain of variola virus. Six macaques were placebo-treated controls, six were tecovirimat-treated beginning at 2 days post-infection, and six were tecovirimat-treated beginning at 4 days post-infection. All macaques were treated daily until day 17. Archived tissues were interrogated using immunohistochemistry, in situ hybridization, immunofluorescence, and electron microscopy. Gross lesions in three placebo-treated animals that succumbed to infection primarily consisted of cutaneous vesicles, pustules, or crusts with lymphadenopathy. The only gross lesions noted at the conclusion of the study in the three surviving placebo-treated and the Day 4 treated animals consisted of resolving cutaneous pox lesions. No gross lesions attributable to poxviral infection were present in the Day 2 treated macaques. Histologic lesions in three placebo-treated macaques that succumbed to infection consisted of proliferative and necrotizing dermatitis with intracytoplasmic inclusion bodies and lymphoid depletion. The only notable histologic lesion in the Day 4 treated macaques was resolving dermatitis; no notable lesions were seen in the Day 2 treated macaques. Variola virus was detected in all three placebo-treated animals that succumbed to infection prior to the study's conclusion by all utilized methods (IHC, ISH, IFA, EM). None of the three placebo-treated animals that survived to the end of the study nor the animals in the two tecovirimat treatment groups showed evidence of variola virus by these methods. Our findings further characterize variola lesions in the macaque model and describe new molecular methods for variola detection.

Differential requirements for FcγR engagement by protective antibodies against Ebola virus.

Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.

Transcriptional Profiling of the Immune Response to Marburg Virus Infection.

UNLABELLED: Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy. IMPORTANCE: Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernible stages of response to infection that correlate with presymptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected, our results suggest that the innate immune response may contribute to overall pathogenesis.

Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

UNLABELLED: Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. IMPORTANCE: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection.

BACKGROUND: Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. RESULTS: In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. CONCLUSIONS: These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.

Efficacy of tecovirimat (ST-246) in nonhuman primates infected with variola virus (Smallpox).

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. Tecovirimat has been shown to be efficacious in preventing lethal orthopoxviral disease in numerous animal models (G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey, et al., J. Virol. 79:13139-13149, 2005; D. C. Quenelle, R. M. Buller, S. Parker, K. A. Keith, D. E. Hruby, et al., Antimicrob. Agents Chemother., 51:689-695, 2007; E. Sbrana, R. Jordan, D. E. Hruby, R. I. Mateo, S. Y. Xiao, et al., Am. J. Trop. Med. Hyg. 76:768-773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 × 10(8) PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.

A novel respiratory model of infection with monkeypox virus in cynomolgus macaques.

Variola, the causative agent of smallpox, and the related monkeypox virus are both select agents that, if purposefully released, would cause public panic and social disruption. For this reason research continues in the areas of animal model and therapeutic development. Orthopoxviruses show a widely varying degree of host specificity, making development of accurate animal models difficult. In this paper, we demonstrate a novel respiratory infection technique that resulted in "classic" orthopox disease in nonhuman primates and takes the field of research one step closer to a better animal model.

In vitro inhibition of monkeypox virus production and spread by Interferon-ß.

BACKGROUND: The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. RESULTS: The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-ß (IFN-ß) for use against monkeypox virus. We found that treatment with human IFN-ß results in a significant decrease in monkeypox virus production and spread in vitro. IFN-ß substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-ß induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. CONCLUSIONS: Our results demonstrate the successful inhibition of monkeypox virus using human IFN-ß and suggest that IFN-ß could potentially serve as a novel safe therapeutic for human monkeypox disease.

Retrospective detection of monkeypox virus in the testes of nonhuman primate survivors.

Close contact through sexual activity has been associated with the spread of monkeypox virus (MPXV) in the ongoing, global 2022 epidemic. However, it remains unclear whether MPXV replicates in the testes or is transmitted via semen to produce an active infection. We carried out a retrospective analysis of MPXV-infected crab-eating macaque archival tissue samples from acute and convalescent phases of infection of clade I or clade II MPXV using immunostaining and RNA in situ hybridization. We detected MPXV in interstitial cells and seminiferous tubules of testes as well as epididymal lumina, which are the sites of sperm production and maturation. We also detected inflammation and necrosis during the acute phase of the disease by histological analysis. Finally, we found that MPXV was cleared from most organs during convalescence, including healed skin lesions, but could be detected for up to 37 d post-exposure in the testes of convalescent macaques. Our findings highlight the potential for sexual transmission of MPXV in humans.

Evaluation of Virulence in Cynomolgus Macaques Using a Virus Preparation Enriched for the Extracellular Form of Monkeypox Virus.

The 2022 global human monkeypox outbreak emphasizes the importance of maintaining poxvirus research, including enriching a basic understanding of animal models for developing and advancing therapeutics and vaccines. Intravenous administration of monkeypox virus in macaques is arguably one of the best animal models for evaluating the efficacy of medical countermeasures. Here we addressed one criticism of the model, a requirement for a high-titer administration of virus, as well as improving our understanding of monkeypox virus pathogenesis. To do so, we infected macaques with a challenge dose containing a characterized inoculum enriched for the extracellular form of monkeypox virus. Although there were some differences between diseases caused by the enriched preparation compared with a relatively similar unpurified preparation, we were unable to reduce the viral input with the enriched preparation and maintain severe disease. We found that inherent factors contained within the serum of nonhuman primate blood affect the stability of the monkeypox extracellular virions. As a first step to study a role of the extracellular form in transmission, we also showed the presence of this form in the oropharyngeal swabs from nonhuman primates exposed to monkeypox virus.

Infection of cynomolgus macaques with a recombinant monkeypox virus encoding green fluorescent protein.

Monkeypox virus (MPXV) causes a vesiculopustular rash illness resembling smallpox in humans and produces a similar disease in nonhuman primates. To enhance the ability of researchers to study experimental MPXV infections, we inserted a gene encoding green fluorescent protein (GFP) into Monkeypox virus Zaire-79. Wild-type and MPXV-GFP replicated with similar kinetics in cell culture and caused a similar disease when injected intravenously into cynomolgus macaques. In MPXV-GFP-infected animals, examination under fluorescent light facilitated the identification of skin lesions during disease development and internal sites of replication at necropsy. MPXV-GFP could improve the quantitative assessment of antiviral therapy and vaccine efficacy.

Delayed Disease in Cynomolgus Macaques Exposed to Ebola Virus by an Intranasal Route.

Ebola virus remains a significant public health concern due to high morbidity and mortality rates during recurrent outbreaks in endemic areas. Therefore, the development of countermeasures against Ebola virus remains a high priority, and requires the availability of appropriate animal models for efficacy evaluations. The most commonly used nonhuman primate models for efficacy evaluations against Ebola virus utilize the intramuscular or aerosol route of exposure. Although clinical disease signs are similar to human cases, disease progression in these models is much more rapid, and this can pose significant hurdles for countermeasure evaluations. The objective of the present study was to evaluate the Ebola virus disease course that arises after cynomolgus macaques are exposed to Ebola virus by a mucosal route (the intranasal route). Two different doses (10 pfu and 100 pfu) and delivery methodologies (drop-wise and mucosal atomization device) were evaluated on this study. Differences in clinical disease between dose and delivery groups were not noted. However, a delayed disease course was identified for approximately half of the animals on study, and this delayed disease was dose and administration method independent. Therefore, it appears that mucosal exposure with Ebola virus results in a disease course in cynomolgus macaques that more accurately replicates that which is documented for human cases. In summary, the data presented support the need for further development of this model as a possible alternative to parenteral and small-particle aerosol models for the study of human Ebola virus disease and for countermeasure evaluations.

Detecting Pathogen Exposure During the Non-symptomatic Incubation Period Using Physiological Data: Proof of Concept in Non-human Primates.

Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices. Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device. Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures - a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation - a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001. Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host's immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.

Natural history of disease in cynomolgus monkeys exposed to Ebola virus Kikwit strain demonstrates the reliability of this non-human primate model for Ebola virus disease.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease.

Selective Targeting of Virus Replication by Proton Pump Inhibitors.

Two proton pump inhibitors, tenatoprazole and esomeprazole, were previously shown to inhibit HIV-1 egress by blocking the interaction between Tsg101, a member of the ESCRT-I complex, and ubiquitin. Here, we deepen our understanding of prazole budding inhibition by studying a range of viruses in the presence of tenatoprazole. Furthermore, we investigate the relationship between the chemistry of prodrug activation and HIV-1 inhibition for diverse prazoles currently on the market. We report that tenatoprazole is capable of inhibiting the replication of members of the enveloped filo, alpha, and herpes virus families but not the flavivirus group and not the non-enveloped poliovirus. Another key finding is that prazole prodrugs must be activated inside the cell, while their rate of activation in vitro correlated to their efficacy in cells. Our study lays the groundwork for future efforts to repurpose prazole-based compounds as antivirals that are both broad-spectrum and selective in nature.

ST-246 antiviral efficacy in a nonhuman primate monkeypox model: determination of the minimal effective dose and human dose justification.

Therapeutics for the treatment of pathogenic orthopoxvirus infections are being sought. In the absence of patients with disease, animal models of orthopoxvirus disease are essential for evaluation of the efficacies of antiviral drugs and establishment of the appropriate dose and duration of human therapy. Infection of nonhuman primates (NHP) by the intravenous injection of monkeypox virus has been used to evaluate a promising therapeutic drug candidate, ST-246. ST-246 administered at 3 days postinfection (which corresponds to the secondary viremia stage of disease) at four different doses (from 100 mg/kg of body weight down to 3 mg/kg) once a day for 14 days was able to offer NHP 100% protection from a lethal infection with monkeypox virus and reduce the viral load and lesion formation. In NHP, the administration of ST-246 at a dose of 10 mg/kg/day for 14 days resulted in levels of blood exposure comparable to the levels attained in humans administered 400 mg in the fed state. These results suggest that administration of an oral dosage of 400 mg once daily for 14 days will be effective for the prevention or treatment of smallpox or monkeypox infections in humans.

Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246.

ST-246, a potent orthopoxvirus egress inhibitor, is safe and effective at preventing disease and death in studies of small-animal models involving challenge by several different pathogenic poxviruses. In this report, the antiviral efficacy of ST-246 in treatment of nonhuman primates infected with variola virus or monkeypox virus was assessed. The data indicate that oral dosing once per day with ST-246 protects animals from poxvirus disease, as measured by reductions in viral load and numbers of lesions and enhancement of survival.

Virulence of Marburg Virus Angola Compared to Mt. Elgon (Musoke) in Macaques: A Pooled Survival Analysis.

Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.