Hiding in the central nervous system: using cerebral organoids to better understand Ebola virus biology
Immunofluorescence image of a brain organoid 41 days after infection with the Ebola virus.
Following infection, the Ebola virus can survive unnoticed in the human body for months or even years, hiding in areas with little immune surveillance like the central nervous system. The danger is that those affected may have an Ebola virus disease relapse or even trigger a new outbreak. Using a cerebral organoid model, researchers at the Bernhard Nocht Institute for Tropical Medicine (BNITM), the German Center for Infection Research (DZIF) and the Icahn School of Medicine at Mount Sinai (ISMMS), together with other collaborators, gained valuable insights into the mechanisms of such Ebola virus persistence. The findings were recently published in Nature Microbiology.
Ebola virus is a filovirus that causes Ebola virus disease, which is a severe and often fatal infection. Even if those affected survive the acute phase of the disease, the virus can remain in the body. Infectious Ebola virus has been detected in semen for months or even a year after infection. The virus can also persist in other immune-privileged organs such as the central nervous system, particularly the brain. Immune-privileged means that the immune system reacts in a weakened and controlled manner in these areas in order to protect sensitive tissue. As a result, it cannot always eliminate the virus completely. This persistent viral presence increases the risk of late inflammatory disease and relapses in individual patients and, albeit rarely, of re-transmission to others.
Cerebral organoids suitable for investigating Ebola persistence
Little is known about the mechanisms that allow Ebola virus to survive long term in its host. Does it persist in tissues or in individual cells? Does it produce new infectious particles? Does it alter its genome to evade detection by our immune system? As research on the human central nervous system is highly complicated, suitable model systems are required instead. This is precisely where the researchers focused their efforts. They successfully used an established cerebral organoid model to perform long-term infection studies. To make these organoids, they stimulated so-called human induced pluripotent stem cells in such a way that they developed into spherical, brain-like structures consisting of various cells of the central nervous system.
“These cerebral organoids enable us to investigate in detail the mechanisms that Ebola virus and other filoviruses use to persist in the human central nervous system. Through experiments in this model system, we can gain insights that help us improve our understanding of the long-term effects of persistence like the severe and sometimes fatal inflammation seen in Ebola virus disease survivors with meningoencephalitis,” explains Dr. Lina Widerspick, first author of the publication and former researcher at the BNITM. She carried out part of the experiments during a research visit to the Integrated Research Facility (IRF)-Frederick of the National Institutes of Health (NIH) in the USA. She is now based at the Bundeswehr Institute of Microbiology in Munich. Moreover, organoids give the unique opportunity to study this phenomenon in a human background rather than an animal model. This may help in re-assessing and optimising treatments like antivirals and further opens avenues to reduce the use of animal models in infectious disease research in the future.
Ebola virus can survive long-term in cerebral organoids
The researchers showed that Ebola virus and other filoviruses, such as Sudan, Reston and Marburg virus, can replicate in cerebral organoids for up to 120 days. They also found that Ebola virus infected various cell types in the cerebral organoids—neurons as well as astrocytes. Microglia, the brain’s immune cells, were also attracted to the site and infected by the virus. Ebola virus was able to spread in the cerebral organoids in two ways: directly from an infected cell to a neighboring cell (cell-to-cell transmission) and by budding from the host cell, which is the classical way the virus spreads. Thus, this represents a ‘productive persistence’, meaning that Ebola virus is not present in an inactive state within cells, but remains infectious.
The cerebral organoids produced pro-inflammatory cytokines, but the immune response was unable to successfully eliminate the virus during the persistent infection. “We observed elevated immune and inflammatory responses in the late stages of cerebral organoid culture. We therefore conclude that a persistent Ebola virus infection in immune-privileged tissues can lead to local inflammation. This observation is consistent with the fact that some Ebola virus disease survivors develop inflammation of the eye, meninges or brain months after infection with Ebola virus”, says Prof. César Muñoz-Fontela, head of the Virus Immunology research group at BNITM and co-last author of the study.
How Ebola adapts to survive
Defective viral genomes are considered a well-known mechanism used by many viruses to suppress their replication. This enables the viruses to survive in the body in an attenuated but long-lasting form. It is also known that the Ebola virus genomes mutate when they replicate for a long time, since their genetic machinery cannot proof-read the genomes as the human machinery would do. The research team has now identified defective viral genomes and particles, and mutations in the Ebola virus genomes in late-stage persistently infected cerebral organoids.
“Many of these mutations had been proposed to reduce or prevent viral replication in naturally occurring infections. Because Ebola virus behaves similarly in this model system to how it does in human infections, this underscores the suitability of our cerebral organoids for investigating filovirus persistence,” explains Prof. Gustavo Palacios, expert on Ebola virus genomics, Professor of Microbiology at ISMMS in New York (USA) and co-last author of the publication. The researchers also identified mutations that have not been described in Ebola virus disease survivors. Further investigations are now needed to determine whether these mutations are causally linked to filovirus persistence.
“Our work in human cerebral organoids highlights the potential of this model system to investigate persistent infections in immune-privileged tissues,” concludes Muñoz-Fontela. “Further studies are now important to investigate the long-term interactions between virus and host, expanding our studies towards less-studied filoviruses like Reston, Taï Forest, Bombali, and Bundibugyo virus, and to deepen our understanding of filoviral persistence mechanisms,” says Muñoz-Fontela, who is also a scientist in the DZIF research area Emerging Infections.
In addition to BNITM, ISMMS, and IRF-Frederick/NIH, the research involved collaboration with investigators from the University Medical Center Hamburg-Eppendorf (UKE), the Leibniz Institute of Virology (LIV), and the Friedrich-Loeffler-Institut—the Federal Research Institute for Animal Health (FLI). Financial support was provided, among others, by the Collaborative Research Center (CRC) 1648 “Emerging Viruses: Pathogenesis, Structure, Immunity” of the German Research Foundation (DFG), the German Federal Institute for Risk Assessment (BfR), and DZIF.
About the Bernhard Nocht Institute for Tropical Medicine (BNITM)
The BNITM is Germany’s largest institution for research, healthcare and teaching in the field of tropical and emerging infectious diseases. Current research priorities include malaria, haemorrhagic fever viruses, neglected tropical diseases (NTDs), immunology, epidemiology and the clinical aspects of tropical infections, as well as the mechanisms of virus transmission by mosquitoes. For handling highly pathogenic viruses and infected insects, the Institute has laboratories of the highest biosafety level (BSL-4) and a biosafety insectarium (BSL-3). In numerous countries of the Global South, the BNITM supports the development of (mobile) laboratory capacities.
The BNITM is the National Reference Centre for Tropical Infectious Agents, a Consultant Laboratory for Bornaviruses, a WHO Collaborating Centre for Arboviruses and Haemorrhagic Fever Viruses, a WHO Collaborating Centre for Behavioral Research in Global Health (BRIGHT), and an institute within the Leibniz Association.
About the Icahn School of Medicine at Mount Sinai (ISMMS)
The Icahn School of Medicine at Mount Sinai is internationally renowned for its outstanding research, educational, and clinical care programs. It is the sole academic partner for the seven member hospitals of the Mount Sinai Health System, one of the largest academic health systems in the United States, providing care to New York City’s large and diverse patient population.
Ranked 11th nationwide in National Institutes of Health (NIH) funding, the Icahn School of Medicine at Mount Sinai is among the 90th percentile of U.S. private medical schools in Sponsored Programs Direct Expenditures per Principal Investigator, according to the Association of American Medical Colleges. More than 6,900 scientists, educators, and clinicians work within and across dozens of academic departments and multidisciplinary institutes with an emphasis on translational research and therapeutics.
Source: Press release of the Bernhard Nocht Institute for Tropical Medicine
