After the ghastly symptoms subside, Ebola may not be done; it may just shift to a clever stealth mode, a new study suggests.

Examining archived tissue samples from infected monkeys, researchers found that Ebola can create a cryptic viral reservoir in certain immune cells and hide in corners of the body where the rest of the immune system has little reach. The study, published this week in Nature Microbiology, echoes the reports from human Ebola survivors who complain of lingering symptoms and complications that researchers have struggled to understand.

Overall, the evidence of persistent infections—which threaten to relapse and spark new outbreaks—adds extra concern for an already alarming pathogen. But researchers are hopeful that the study also provides a way forward for research into defeating this stage of infection.

“Understanding the molecular–virological mechanisms of [Ebola virus] persistence is of paramount importance, including the conditions that favour persistence and reactivation and the time frame in which persistence may occur,” the authors conclude. “Our study clarifies that a robust rhesus monkey model for [Ebola virus] persistence could be developed.”

The authors, led by Xiankun Zeng and colleagues at the US Army Medical Research Institute of Infectious Diseases (USAMRIID), began focusing on Ebola persistence in the wake of the 2013 to 2016 outbreak in Western Africa. That outbreak had more Ebola survivors than ever—17,323 (60 percent) of the outbreak’s 28,646 reported patients worldwide survived.

Though survivors were once thought to be fully recovered and healthy, about 50 percent go on to complain of eye and vision problems. Others complain of muscle and joint pain, hearing loss, and neurological problems. Some survivors have Ebola still lurking in seminal fluid, and a couple have passed the infection on to a partner. Just one new case of Ebola can start another outbreak cluster, the researchers warn.

Hide & seek

Despite the accounts, this collective “post-Ebola syndrome” is poorly understood. It has been unclear if Ebola is causing low-grade infections, goes dormant for a time, or if residual damage and inflammation is causing some of the symptoms. Sometimes researchers can find Ebola still lurking, such as in one case of a survivor with eye inflammation. But other times, researchers can’t, such as a recent case study of seven survivors with neurological complications who all turned up negative for Ebola RNA in their cerebrospinal fluid.

To figure out what might be going on, Zeng and his colleagues looked at archived tissue samples from a past study involving 112 rhesus monkeys. The monkeys had all survived 43 days after researchers exposed them to Ebola and were given either a non-vaccine medical treatment or no treatment. Then they were euthanized at the end of the 43-day study. Zeng and his team looked for Ebola in the monkeys’ eye, testicle, brain, liver, spleen, and lymph node tissue samples. Though the 112 survivor monkeys had no sign of Ebola in their blood at the time of death, 11 had detectable Ebola RNA in at least one tissue sample. The only types of tissue that tested positive were those from the eye, testicle, and brain—matching the suspected sites of persistent infections in human survivors.

To try to understand how the infection may have progressed to be persistent in those 11 monkeys, the researchers turned to tissue samples from animals that had fatal cases. They got eye tissue samples from 24 monkeys that succumbed to acute disease quickly after exposure (five to 11 days), plus samples from 14 monkeys with delayed Ebola disease (16 to 24 days post-exposure).

Digging in

Looking along the three groups, the researchers noted that the longer out from the Ebola exposure, the deeper the virus penetrated the layers of tissue and structures in the animals’ eyes. In the animals that died quickly (five to 11 days), the virus mainly replicated in blood vessels. In those that had a delayed death (16 to 24 days), the virus had spread into surrounding tissue. And in the survivors, the virus had reached deep into the “immune privileged” site. These are sites in which the immune system is known to be less active when protecting certain tissue from potentially harmful immune responses. Sites of immune privilege include the brain and testes, as well as the eye. Fitting with this finding, the researchers noted that the virus had damaged the blood-brain barrier in survivors, as well.

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Along with deeply infected tissue among the survivors’ samples, researchers found patterns of inflammation and Ebola virus in one particular type of immune cell—CD68+ macrophages. These cells are normally deployed to feast on pathogens at the site of an infection. But they instead seem to provide a key reservoir for the virus, the researchers reported.

Though the study offers clear evidence of persistent Ebola infections in nonhuman primates, researchers will need more data to confirm that’s going on in humans with post-Ebola syndrome. And beyond that, a lot more questions still need to be addressed, such as how long the virus persists, in what conditions, and how it might reignite an infection and spread.

To answer some of those questions, researchers will need an animal model to study persistence. Most animal models of Ebola infections result in nearly 100-percent fatalities in short order—nixing the chance of developing persistent infections. But Zeng and his colleagues are hopeful that their data can lead researchers to a model of persistence using medically treated rhesus monkeys. The researchers say they’ve already begun work on developing such a model.

Nature Microbiology, 2017. DOI: 10.1038/nmicrobiol.2017.113 (About DOIs).