In early 2009, Joe DeRisi — co-president of the Chan Zuckerberg Biohub and chair of biochemistry and biophysics at UC San Francisco — unearthed a handwritten letter from the bottom of an old stack of mail. In the note, long-time San Jose resident and snake enthusiast, Taryn Hook, begged DeRisi to investigate a mysterious and incurable neurological disease that was killing her pet snake, Larry. This so-called “inclusion body disease” in snakes isn’t pretty. First, the snakes throw up, or they just stop eating altogether. Later, the disease attacks the snakes’ brains, until eventually the infected snakes wave their heads uncontrollably, get stuck on their backs, or tie themselves in knots. 

Years earlier, during his graduate studies, DeRisi streamlined a diminutive but powerful diagnostic tool that houses tens of thousands of microscopic, sticky DNA barcodes, or “probes,” each probe corresponding to a unique stretch of DNA. The tool, called a DNA microarray or a DNA ‘Chip,’ allowed him to investigate which genes got switched on or off inside cancer cells, revolutionizing the study of cancer biology. But it was DeRisi’s later work that would lead him to the investigation of deadly viruses. While studying the regulation of human genes in a kind of skin cancer caused by infection with a herpesvirus, DeRisi used the same human DNA barcoding technology to study genes from the herpesvirus itself. And from there, it was quick work to develop a DNA chip that could detect more than one virus, with early models detecting over 20,000 distinct species. Under magnification, the chip looks like a plaid swatch lightly dusted with fluorescent sprinkles, each sprinkle representing a positive “hit” of viral DNA. Even in the early days, the thumb-sized “virochip” was a massive genetic database, able to diagnose known viruses and, by inference, identify new ones. And in 2009, thanks to Tarryn Hook and her ill-fated reptilian friend, DeRisi had an intriguing new illness to investigate.

Scientists have known about inclusion body disease for a while, but nobody knew what caused it. Defined by a distinctive build-up of inclusion bodies – or protein clumps – inside cells, the disease was thought to be transmitted by an infectious agent like a virus. In every case, said DeRisi, “If you put a healthy snake in with a sick snake, they both got it. The healthy one became ill.” DeRisi reasoned that if he could compare the DNA of a healthy snake to the DNA of a snake with inclusion body disease, powerful computers could filter out the snake DNA and reveal snippets of non-snake DNA, presumably belonging to whatever it was that was making the snakes sick.

And it worked. DeRisi and his colleagues zeroed in on a new member of the Arenavirus family. Other Arenaviruses infect people, and represent the largest family of viruses known to cause hemorrhagic fevers, but this was the very first time an Arenavirus had been discovered in reptiles.  Even more remarkable, though, was a piece of the Arenavirus called a glycoprotein. A virus glycoprotein is like an access badge for the virus to get into the secure insides of a cell – an essential component of the entry process. Typically, viruses from the same family have similar glycoproteins, except in this case, it was clear that this particular glycoprotein was an interloper. According to DeRisi, the glycoprotein wasn’t from the Arenavirus lineage. “It was from filovirus. It was from Ebola.”

On the microscopic level, Ebola is a long-limbed ampersand of death, while Arenavirus trends to the rotund, resembling a pufferfish or lychee fruit. But these divergent shapes reflect their distant phylogenetic relationship…so how did the snake Arenavirus end up with Ebola’s glycoprotein? This kind of switcheroo happens occasionally in virus evolution – sometimes viruses cross paths in a shared host and swap genes. To be clear, this hybrid virus is not Ebola-in-disguise. It’s an Arenavirus that somehow managed to steal Ebola’s access badge. It’s deadly for snakes, but harmless to humans because – like in every heist movie – breaking into a bank is just the first step. You still need all the right tools to steal the gold or blow up the safe. And this particular Arenavirus just doesn’t have the right set of tools to hurt us.  

But what it did seem to indicate is that if this Arenavirus could infect snakes – with Ebola’s access badge – then there was a pretty good chance that Ebola can get into snakes, too.

“To put it another way, how do we know [Ebola’s] not in reptiles right now?” said DeRisi.

A Perfect Host

Ebola’s less famous cousin, Marburg Virus, comes from bats, and the evidence for a Marburg-bat-reservoir is decisive. Stories of cave tourists contracting Marburg led researchers to explore the connection between the caves’ winged inhabitants and the deadly virus. It turned out Marburg could be grown, or “cultured,” easily in bat cells without killing them and it has been isolated repeatedly from wild bats – a great one-two punch for infectious disease epidemiology. So Marburg, said DeRisi, “…definitely a bat virus.” This evidence has led many to assume that bats must also be the natural reservoir for Ebola, which hails from the same family of filamentous viruses as Marburg, called filoviridae. But, asked DeRisi, “what’s the evidence that ebola virus is actually in bats?”

Turns out – not much.  In 2003, during an outbreak in Gabon, scientists discovered fragments of Ebola’s genes in fruit bats, but were unable to detect the whole virus, and after an exhaustive search, found no convincing evidence of active or recent ebola infection in the bats. Ebola is also known to infect other mammals, including pigs, chimpanzees, and dogs, but these species are poor candidates to be Ebola’s long-term host. One big requirement for identifying a natural reservoir species is that the infection persists in the population and is asymptomatic. However, in these mammals, Ebola infection tends to be either short-lived, moderately symptomatic, or deadly.

But…reptiles? Typically, epidemiologists don’t think of reptiles while working to identify reservoir species. “Why would you? They’re so alien,” quipped DeRisi. And so far, there are no known examples of deadly viruses that can jump between reptiles and humans.

Except in folklore.

A Witch Walks into a Village

During the 2014 Ebola outbreak in West Africa that killed over 11,000 people, a story made its way from village to village, with a prelude that sounds like the set-up for a bad joke: “A witch walks into a village.” In every version of the story, “she’s carrying this big bag” says DeRisi, and, before disappearing into the African bush, the witch deposits her bag with a warning to the curious townsfolk to leave it untouched (in other interpretations, she dies). The penultimate scene plays out with a sub-Saharan version of the ill-fated Pandora and her jar of evils: intrepid villager peers into witch’s bag, sees a snake (!), and dies 48 hours later. The coda is no less grim: a few days after the unwitting release of the deadly virus, most of the other villagers have succumbed.

In 2014, on their Goats and Soda series, NPR dismissed the legend of the witch and her snake with a single (snarky) headline: “Rumor patrol: No, a snake in a bag did not cause Ebola.” When officials are working swiftly to contain a virus spread primarily through human contact, these snake bag narratives could be dangerous distractions, claimed the author. And there is a crisp sort of logic to this – far more people get Ebola from other people – not from snakes. But we still have no idea where ebola lurks between outbreaks.

Enter DeRisi.

Confronted with the seductive mystery of the snake arenavirus and its hijacked Ebola glycoprotein, DeRisi needed help. Specifically, he needed a facility that could safely handle Ebola, so he turned to Jens Kuhn, PhD., head of virology at the NIH’s new Biosafety Level-4 (BSL4), a type of laboratory used to study only the most dangerous pathogens – like Ebola, or other hemorrhagic fever viruses.

And “we did this really crazy experiment,” said DeRisi. The experiment was simple: harvest cells from a snake and put Ebola on them. If the virus failed to infect the snake cells, then DeRisi was probably barking up the wrong tree. On the other hand, a positive result still did not mean that he found the reservoir…it just meant that it was plausible.

His colleagues at the BSL-4 dropped a whiff of ebola onto the snake cells and then onto some human cells in a Petri dish nearby. Twenty-four hours later, all of the human cells were dead. “The plate’s decimated. That’s what Ebola does,” said DeRisi. Then they turned to the other dish and, to their excitement, the snake cells looked completely healthy.

When they checked for new virus production, the results blew them away. The whole dish was productively infected, with up to a billion new Ebola virus particles released into the fluid around the cells. DeRisi had just established a definitive link between Ebola and reptiles. But he still wanted to know exactly how easy it was for the virus to cross the species barrier from humans to snakes.

We’re back to our virus access badge problem. Many viruses deadly to humans, like SARS, develop in a non-human host and make the leap to humans after a handful of lucky mutations tweak the structure of proteins critical for invasion – a series of serendipitous modifications to the access badge. DeRisi and his colleagues were struck by how quickly Ebola thrived in the snake cells. They expected that — if it worked at all — it should take much longer. Time enough for the human form of the virus to tinker with its access badge and make the jump into snakes.

But what if Ebola didn’t require any changes to jump between humans and snakes?

DeRisi sequenced the DNA of the “human” ebola and the “snake” ebola, and discovered that the viruses were identical. “There are no required amino acid changes to be perfectly fine in a reptile cell.” DeRisi’s team had just discovered that Ebola can switch seamlessly between human cells and reptile cells without a single genetic change.

And now the snake-in-a-bag story starts to feel shockingly plausible. Snakes are eaten as bushmeat in Guinea and Sierra Leone, so transmission could come from eating, bites, or shedding. “There’s a million ways that you can spread a virus,” said DeRisi.

The Next Experiment

So, what happens now? While it slogs through review at a top scientific journal, DeRisi’s story is already up in BioRxiv, the most popular preprint server for biologists. But until his story is fully vetted, DeRisi remains understandably reticent. “I don’t talk about the Ebola project much. You know why? Because we would be accused of being crazy.”

It’s an exciting twist to a decades-old story, and after the recent Ebola outbreak in West Africa, the stakes have never been higher. The next experiment – before anybody goes off on an expensive expedition looking for infected snakes – is to challenge live snakes in a lab environment with Ebola. “I think if a python can be productively infected, that would be strong evidence that we should go look. Now, who’s going to fund that? How are we going to get that done? I have no idea,” said DeRisi.

And maybe the legends are true – maybe  a woman really did have a bag with a snake inside, and maybe that snake really did carry Ebola.

We could crack this case soon – just don’t sign me up for that expedition.

 

References

Click to access mBio-2012-Stenglein-sm.pdf

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