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Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys

Nature Microbiology volume 2, Article number: 17113 (2017) Cite this article

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Abstract

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013–2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.

Ebola virus disease (EVD) is a severe human viral haemorrhagic fever caused by infection with either Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV) or Taï Forest virus (TAFV)1. From 2013 to 2016, an EBOV-caused EVD outbreak in Western Africa led to a total of 28,646 cases and 11,323 deaths worldwide (case-fatality rate of 40%)2. The large number of EVD survivors revealed previously underestimated sequelae of EBOV infection or ‘post-Ebola (virus disease) syndrome’. This syndrome includes arthralgia, cognitive impairment, headaches, hearing loss and myalgia3–6. About 50% of surveyed survivors of this outbreak complained of various ocular complications such as blurry vision, pain and discomfort. One-fifth of these survivors were diagnosed with uveitis4,7–13. In one uveitis case, EBOV was isolated from the eye12. Another EVD survivor developed meningoencephalitis more than 9 months after convalescence, and EBOV could be isolated from the patient's cerebrospinal fluid9.

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Figure 1: Detection of genomic EBOV RNA in the eyes of rhesus monkey survivors by in situ hybridization.
Figure 2: Ocular macrophages of survivors express EBOV GP1,2 antigen.
Figure 3: Persistent EBOV infection causes uveitis, retinitis and vitritis accompanied by reactive gliosis.
Figure 4: Ebola virus persistence in the brain of a rhesus monkey survivor.
Figure 5: EBOV persistently infects the epididymis of a rhesus monkey survivor.
Figure 6: EBOV replicates in the eyes, epididymides and brains of rhesus monkey survivors.

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Acknowledgements

The authors thank S. Lockett and K. Peifley at the Optical Microscopy and Analysis Laboratory, National Cancer Institute at Frederick (Fort Detrick, Frederick, MD, USA) for use of their confocal microscope. The authors thank L. Bollinger and J. Wada (NIH/NIAID Integrated Research Facility at Fort Detrick, Frederick, MD, USA) for critically editing the manuscript and figure preparation, respectively. The authors thank W. Discher and J. Braun at United States Army Medical Research Institute of Infectious Diseases (USAMRIID, Fort Detrick, Frederick, MD, USA) for the eye diagram and M. Kortepeter, A. Cardile, C. Shaia and A. Anderson for their critical reading of the manuscript. Work at USAMRIID was funded by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) and the Medical Countermeasure Systems (MCS) of the Joint Program Executive Office for Chemical and Biological Defense (JPEO-CBD). This work was supported in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under contract no. HHSN272200700016I. A subcontractor to Battelle Memorial Institute who performed this work is: J.H.K., an employee of Tunnell Government Services, Inc. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the US Department of the Army, the US Department of Defense, the US Department of Health and Human Services, or of the institutions and companies affiliated with the authors. In no event shall any of these entities have any responsibility or liability for any use, misuse, inability to use, or reliance upon the information contained herein. The US departments do not endorse any products or commercial services mentioned in this publication.

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  1. United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, 21702, Maryland, USA

    Xiankun Zeng, Candace D. Blancett, Keith A. Koistinen, Christopher W. Schellhase, Jeremy J. Bearss, Sheli R. Radoshitzky, Shelley P. Honnold, Taylor B. Chance, Travis K. Warren, Jeffrey W. Froude, Kathleen A. Cashman, John M. Dye, Sina Bavari, Gustavo Palacios & Mei G. Sun

  2. Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, B-8200 Research Plaza, Frederick, 21702, Maryland, USA

    Jens H. Kuhn

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Contributions

X.Z. conceived and designed the experiments. X.Z. and C.D.B. performed the in situ hybridization. X.Z. performed the immunofluorescence staining and confocal imaging. K.A.K., C.W.S., J.J.B., S.P.H. and T.B.C. performed histopathology. T.K.W., J.W.F., K.A.C., J.M.D. and S.B. provided experimental materials. X.Z., S.R.R., G.P. and J.H.K. interpreted the data and wrote the manuscript. S.B. and M.G.S. coordinated and oversaw the study.

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Correspondence to Xiankun Zeng.

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Supplementary Tables 1–4, Supplementary Figures 1–3. (PDF 1112 kb)

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Zeng, X., Blancett, C., Koistinen, K. et al. Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys. Nat Microbiol 2, 17113 (2017). https://doi.org/10.1038/nmicrobiol.2017.113

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