Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others


  1. Kuhn, J. H. in Harrison's Principles of Internal Medicine Vol. 2 (eds Kasper, D. L. et al.) 1323–1329 (McGraw-Hill Education, 2015).

    Google Scholar 

  2. Ebola Situation Report – 30 March 2016 (WHO, 2016);

  3. Burki, T. K. Post-Ebola syndrome. Lancet Infect. Dis. 16, 780–781 (2016).

    Article  PubMed  Google Scholar 

  4. Epstein, L., Wong, K. K., Kallen, A. J. & Uyeki, T. M. Post-Ebola signs and symptoms in U.S. survivors. N. Engl. J. Med. 373, 2484–2486 (2015).

    Article  PubMed  Google Scholar 

  5. Scott, J. T. et al. Post-Ebola syndrome, Sierra Leone. Emerg. Infect. Dis. 22, 641–646 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Carod-Artal, F. J. Post-Ebolavirus disease syndrome: what do we know? Expert Rev. Anti. Infect. Ther. 13, 1185–1187 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Mattia, J. G. et al. Early clinical sequelae of Ebola virus disease in Sierra Leone: a cross-sectional study. Lancet Infect. Dis. 16, 331–338 (2016).

    Article  PubMed  Google Scholar 

  8. Deen, G. F. et al. Ebola RNA persistence in semen of Ebola virus disease survivors—preliminary report. N. Engl. J. Med. (2015).

  9. Jacobs, M. et al. Late Ebola virus relapse causing meningoencephalitis: a case report. Lancet 388, 498–503 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Soka, M. J. et al. Prevention of sexual transmission of Ebola in Liberia through a national semen testing and counselling programme for survivors: an analysis of Ebola virus RNA results and behavioural data. Lancet Global Health 4, e736–e743 (2016).

    Article  PubMed  Google Scholar 

  11. Sow, M. S. et al. New evidence of long-lasting persistence of Ebola virus genetic material in semen of survivors. J. Infect. Dis. 214, 1475–1476 (2016).

    Article  PubMed  Google Scholar 

  12. Varkey, J. B. et al. Persistence of Ebola virus in ocular fluid during convalescence. N. Engl. J. Med. 372, 2423–2427 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Uyeki, T. M. et al. Ebola virus persistence in semen of male survivors. Clin. Infect. Dis. 62, 1552–1555 (2016).

    Article  PubMed  Google Scholar 

  14. Diallo, B. et al. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days. Clin. Infect. Dis. 63, 1353–1356 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Christie, A. et al. Possible sexual transmission of Ebola virus—Liberia, 2015. MMWR 64, 479–481 (2015).

    PubMed  PubMed Central  Google Scholar 

  16. Mate, S. E. et al. Molecular evidence of sexual transmission of Ebola virus. N. Engl. J. Med. 373, 2448–2454 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Blackley, D. J. et al. Reduced evolutionary rate in reemerged Ebola virus transmission chains. Sci. Adv. 2, e1600378 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Arias, A. et al. Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases. Virus Evol. 2, vew016 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Statement on the 9th Meeting of the IHR Emergency Committee Regarding the Ebola Outbreak in West Africa (WHO, 2016);

  20. Shurtleff, A. C. & Bavari, S. Animal models for ebolavirus countermeasures discovery: what defines a useful model? Expert Opin. Drug Discov. 10, 685–702 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Geisbert, T. W. et al. Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet 362, 1953–1958 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Larsen, T. et al. Pathologic findings associated with delayed death in nonhuman primates experimentally infected with Zaire Ebola virus. J. Infect. Dis. 196(suppl. 2), S323–S328 (2007).

    Article  PubMed  Google Scholar 

  23. Warren, T. K. et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 531, 381–385 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Alves, D. A. et al. Necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an Ebola virus-infected rhesus macaque (Macaca mulatta) with apparent recovery and a delayed time of death. J. Infect. Dis. 213, 57–60 (2016).

    Article  PubMed  Google Scholar 

  25. Geisbert, T. W. et al. Pathogenesis of Ebola hemorrhagic fever in primate models. Am. J. Pathol. 163, 2371–2382 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schnittler, H.-J. & Feldmann, H. Marburg and Ebola hemorrhagic fevers: does the primary course of infection depend on the accessibility of organ-specific macrophages? Clin. Infect. Dis. 27, 404–406 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. MacLaren, R. E. Development and role of retinal glia in regeneration of ganglion cells following retinal injury. Br. J. Ophthalmol. 80, 458–464 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dyer, M. A. & Cepko, C. L. Control of Muller glial cell proliferation and activation following retinal injury. Nat. Neurosci. 3, 873–880 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Martines, R. B., Ng, D. L., Greer, P. W., Rollin, P. E. & Zaki, S. R. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses. J. Pathol. 235, 153–174 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Rodriguez, L. L. et al. Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995. J. Infect. Dis. 179(suppl. 1), S170–S176 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Carroll, M. W. et al. Temporal and spatial analysis of the 2014–2015 Ebola virus outbreak in West Africa. Nature 524, 97–101 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Nakayama, E. & Saijo, M. Animal models for Ebola and Marburg virus infections. Front. Microbiol. 4, 267 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Sanchez, A., Kiley, M. P., Holloway, B. P. & Auperin, D. D. Sequence analysis of the Ebola virus genome: organization, genetic elements, and comparison with the genome of Marburg virus. Virus Res. 29, 215–240 (1993).

    Article  CAS  PubMed  Google Scholar 

  34. Mühlberger, E., Weik, M., Volchov, V. E., Klenk, H.-D. & Becker, S. Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73, 2333–2342 (1999).

    PubMed  PubMed Central  Google Scholar 

  35. Wang, F. et al. RNAscope a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wilson, J. A. et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664–1666 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Xiankun Zeng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing