Can the Egyptian fruit bat’s unusual genome show us how to fight deadly Marburg virus?

The Egyptian fruit bat's immune system enables it to peacefully co-exist with Marburg virus, which can cause a swiftly deadly infection in humans. Although Marburg virus disease affects only a few dozen or hundred people a year, the case:fatality ratio in the scattered outbreaks ranges from 50% to 100%. A recent paper in Cell that explores the bat's genome reveals how its immune system may prevent the virus from harming the flying mammals, which may hold clues for preventing or treating the infection in humans.

The Egyptian fruit bat Rousettus aegyptiacus lives in groups of 1,000 to more than 100,000 in caves and mines in many places in Africa. The bat weighs less than half a pound and is only half a foot long, but the wings stretch to two feet. Males are distinguishable by their large scrotums, and the bats are highly vocal.

Marburg virus, and the related Ebola virus, are filoviruses. They have a single strand of RNA as their genetic material.

People are infected with Marburg virus when they touch bat excrement, body fluids, or tissue, and can transmit the infection to other people through direct contact. After an incubation period of 2 to 21 days, the illness starts suddenly with high fever, excruciating headache, and aches and pains. Day three brings severe watery diarrhea, abdominal cramps, and nausea and vomiting.

A rash may appear during the first week. But the patient might not live that long because this is also when the gums, nose, vagina, and rectum bleed, along with bloody vomit, which is why Marburg virus disease was formerly called Marburg hemorrhagic fever. Trying to insert an IV to counteract the fluid loss triggers even more bleeding. Meanwhile, cerebral edema leads to confusion, irritability and aggressive behavior. The bleeding may plunge the patient into shock, leading to death.

In survivors, Marburg virus can remain in body parts shielded from the immune system, like the testicles and inside the eyes. In women infected while pregnant the virus can nestle in the placenta, amniotic fluid and fetus, and persist in breast milk.

Investigating caves and mines

The first recorded cases of the disease were from 1967, with 23 cases in Marburg, Germany, 6 cases in Frankfurt, and 2 in Belgrade, Serbia. Twenty-six of the cases were lab workers who had contracted the viral infection from African green monkeys from Uganda in efforts to develop a polio vaccine. Nine of the 26 people died. Over the years a few cases appeared in Angola, the Democratic Republic of the Congo, Kenya, South Africa, and Uganda.

But the reservoir – the healthy species that harbors the pathogen, enabling it to “spill over” into a vulnerable species like our own – remained a mystery. Bats were high on the list of suspects, though, because they were known to carry Ebola virus RNA.

Then an outbreak in 2007 pointed to the Egyptian fruit bat. Four men mining for galena and gold in Kitaka Cave in Uganda fell ill, and one of them died. Such caves typically house thousands of the bats, which, disturbed by the miners, flutter about, leaving sticky guano everywhere. Kitaka Cave had about 100,000 bats.

A team from the Centers for Disease Control and Prevention, the World Health Organization, and the National Institute for Communicable Diseases in South Africa investigated the poop-filled mine. They removed about 400 bats and came back 9 months later for 200 more, to see if Marburg virus, if it was there, had persisted. The investigators also captured and put necklaces on 1,329 bats, then released them. Would the marked bats resurface elsewhere, and if so, how far away? Such a low-tech approach would turn out to reveal valuable information.

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When the team sent bat blood to the CDC, the researchers there identified antibodies against the virus and bits of viral RNA in 31 of the bats – about 5% – and isolated live virus from 5 bats. That was the first look at Marburg.

The five viral genomes found in the bats were incredibly diverse, which meant the bats had been harboring the viruses for a long time, enough to have accumulated mutations. In addition, the Marburg virus genomes collected from the bats and viruses found in the miners, closely matched.

Marburg virus

The researchers published a key paper in 2009 in PLOS Pathogens. But while they were analyzing their data and writing the paper introducing the Marburg reservoir, two tourists encountered the virus. Only one lived to tell the tale.

In early 2008 Astrid Joosten, from the Netherlands, vacationed with her husband in Uganda. They took a side trip from seeing mountain gorillas to visit Python Cave, whose reptilian namesakes grow huge from their abundant fruit bat meals.

The couple only explored the guano-sticky snake cave for about 10 minutes, but in that time Astrid must have touched some of the excrement that festooned all the surfaces, her husband recalled. She either had a cut on her hand where the virus entered, or touched her hand to her mouth.

However infection happened, Astrid developed a raging fever 13 days later, and then as her organs failed, she bled. Yet her husband, who’d scrabbled through the same rocky cave and had even kissed his dying wife in the hospital, was ok. David Quammen, in his excellent book Spillover, describes another case, of an American who visited Python Cave months before Astrid and developed a never-diagnosed mysterious illness that would turn out to have been Marburg. She survived.

Python Cave is only about 30 miles from Kitaka cave, where the gold miners were infected. After Astrid’s death, a research team discovered a beaded collar on the guano-encrusted floor of Python Cave. It had clearly come from Kitaka Cave, where some of the bat residents had been outfitted with the necklaces. So the bats can fly pretty far – and perhaps even hop across the continent, as some 5 percent of them, albeit healthy, shed the deadly (to us) virus.

The most recent outbreak was 3 cases in Uganda in late 2017. A man in his thirties, a game hunter, lived near a bat-infected cave and spread the infection to two others. All three people died.

Probing the Egyptian fruit bat genome

The sequencing of the bat's genome began when Jonathan Towner, a researcher with the CDC's Viral Special Pathogens Branch, collected healthy bats from Python Cave and took them to investigators at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). They performed a quick sequencing and sent the findings to Stephanie Pavlovich, an MD/PhD student in the laboratory of Thomas Kepler, a professor at the National Emerging Infectious Diseases Laboratories at Boston University.

Pavlovich, who is first author, wanted to take a closer look than the previous sequenced bat genomes – 14 species done to date – which are derived from many short DNA pieces, or "reads," which  misses some parts. So she resequenced the genome using longer reads – a little like reading an entire novel rather than excerpts. It took two years.

The team compared the new-and-improved bat genome sequence to those of other bats, guinea pigs, and humans, to distinguish genes common to mammals from those that might provide the Egyptian fruit bat with its protection from Marburg virus. Gene variants unique to the bats would be signs of positive selection – a mutation that provided an advantage, and so has withstood the test of natural selection. It persists.

Detecting signs of positive selection "allowed us to find a number of genes that were evolving at a faster rate in this bat, and then also a number of gene families that were much larger than we expected," said Pavlovich in a news release. "We were looking for gene families that had grown either much larger or much smaller than was expected, given the evolutionary history of this bat," she added. The full sequencing plus other techniques also revealed repeated sequences, which are associated with genes that encode proteins important in the innate immune response.

Two gene families emerged as expanded in the Egyptian fruit bat: those that encode molecules (ASG15, IFNAR1, type 1 interferon receptor, and SIKE1) that dampen the effects of type 1 interferon, which would alert the immune system to virally-infected cells. And the bats have unusual combinations of the components of natural killer (NK) cell receptors. NK cells alert the immune system to the presence of virally-infected cells, so that other molecules and cells can destroy them.

Somehow, the nuances of the bat's immune response allow neither rampant inflammation to quell the virus, nor let it melt the body into a puddle of blood and tissue, as it does a human. The researchers write that “certain key components of the immune system in bats have co-evolved with viruses toward a state of respective tolerance and avirulence,” which is what enables the animals to live peaceably with Marburg.

Dr. Kepler terms the bat's measured immune response "soft protection." The animals seem to have a finer control over the composition of the key NK receptors than do other mammals. "The bat may be assuaging the virus for a short period of time, trying to prevent the virus' growth without making a full-on attack. There's something really interesting going on here," he added.

And so the bat’s immune system doesn’t fight off Marburg; it just ignores the viral infection. The new work points drug developers to screen or synthesize compounds that might tweak the actions of the appropriate interferons and natural killer cell receptors to also blind human cells to the deadly (to us) virus.

Ricki Lewis is the GLP’s senior contributing writer focusing on gene therapy and gene editing. She has a PhD in genetics and is a genetic counselor, science writer and author of The Forever Fix: Gene Therapy and the Boy Who Saved It, the only popular book about gene therapy. BIO. Follow her at her website or Twitter @rickilewis.

 

 

 

 

 

 

 

 

 

 

 

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