Metro

As clock ticks, Cambridge lab hunts for Ebola answers

At 1 p.m. on a Saturday in mid-November, Stephen Gire arrived at the airport in New Orleans, headed back to Boston. The 32-year-old research scientist from Harvard and the Broad Institute was holding a Styrofoam box that he intended to carry on board until he discovered it was too large to fit in the overhead bin. He would have to check it, and that made him extremely nervous.

He wasn’t worried about the contents, only the reaction TSA screeners would surely have if they cracked it open without him there to explain what it was or protect the contents.

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Inside, packed in dry ice, was a smaller box, labeled “Ebola.”

Gire knew the 425 samples of Ebola virus he was bringing back to Boston weren’t hazardous. Before they had been shipped from West Africa, the samples had been treated with a sort of scientist’s Lysol that had rendered the deadly virus harmless.

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But any delay in getting the samples to the lab seemed unacceptable. The epidemic rages on, and bioscience is desperately racing to catch up.

The drops of serum in the box could hold clues to stopping a killer. Drawn from the blood of the sickest people in one of the poorest corners of the world, the samples were destined for the Broad Institute’s massive genomics platform in Cambridge. There, Gire and other scientists would use the most sophisticated genetic machinery known to sequence the Ebola virus and determine how it might be attacked — and if new mutations could make it even more perilous.

Cambridge, MA., 11/24/14, Scientists at the Broad Institute----this is Stephen Gire---- lost three of their partner doctors in Africa this summer. They are working to sequence the ebola virus. Section: Met

Suzanne Kreiter/Globe staff

Stephen Gire returned from Sierre Leone with 425 samples of Ebola virus taken from infected patients.

It is a terrifying possibility. In West Africa in the last year, Ebola killed about 8,000 people and infected more than 20,000 — eight times the number infected in all previous Ebola outbreaks combined. Gire knew those statistics could prove to be only a prelude if the virus turned out to be mutating in a way that made it more elusive to the diagnostic tools in use, and more resistant to the treatments and vaccines in development. That meant lives were hanging in the balance as the Cambridge team searched for answers, a search that couldn’t begin until the samples got safely to Kendall Square.

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Only when his white box came down the baggage claim conveyer belt at Logan, apparently unharmed, did Gire fully exhale. He took the samples straight to Cambridge, to the lab run by Pardis Sabeti, a noted computational geneticist at Harvard and the Broad Institute. There, he stored them in a minus-80 degree refrigerator to await sequencing — and, the world hopes, answers.

From lab to the front lines

Cambridge, MA., 12/05/14, For Neil Swidey story on the hunt for the ebola virus at the Broad Institute. Kendra West, Process Development Associate at the Broad Institute's Genomics Platform, works with the virus in the lab. She is checking the re-agent pumps in the sequencer. Section: Capitol

Suzanne Kreiter/Globe staff

At the Broad Institute’s genomics platform in Cambridge, where process development associate Kendra West worked with samples that were being sequenced, researchers hope to find clues in Ebola’s genetic code.

The Sabeti lab is no newcomer to the Ebola fight.

The team has been working in West Africa for the last half-dozen years on the fight against Ebola and a more common but less fatal viral disease called Lassa fever.

In March of last year, Gire traveled to Sierra Leone, to work with the Sabeti team’s African colleagues at a government lab and hospital in Kenema, the country’s third-largest city. He specializes in improving the efficiency of lab operations, drawing on skills honed in a previous career as a chef running a busy restaurant kitchen in Colorado. (Invited to audition for “Top Chef,” he demurred, having found his vocation in infectious disease research.)

In one unforgettable shift in Sierra Leone, Gire tested samples for Ebola and Lassa for nearly four hours straight, wearing bulky protective gear in 85-degree heat. At one point, when he looked down at his notebook, he was alarmed. Sweating profusely and dizzy, he couldn’t make sense of what he had just written. Sensing he was severely dehydrated and had become a danger to himself and others, he hurried out of the lab, “de-conned” out of his protective suit, and spent the next hour drinking water.

That was before the devastating Ebola outbreak had crossed over from Guinea and Liberia into Sierra Leone. But in late May, after the Kenema lab sent a suspected sample to Cambridge, the Sabeti team confirmed the worst fears of everyone in Sierra Leone. Ebola was now within its borders.

That sparked an extraordinary drive in June to capture samples from the vast majority of people in Sierra Leone infected with Ebola during the first three weeks of the virus’s entry into the country. Those 99 samples were sent to Cambridge, where Sabeti led her 22-member team in a round-the-clock effort to sequence and analyze them, sketching out the genomic portrait of the outbreak.

By July, Gire and colleague Nathan Yozwiak, a lab project manager, were on a plane back to Sierra Leone, to support their African colleagues in the lab and advise government officials who were desperately trying to manage the outbreak. They briefed the officials, in near real time, based on what their colleagues in Cambridge were finding.

What they shared had an immediate impact. At the time, the government was aggressively pushing a public health campaign to warn citizens about the danger of eating bushmeat, flesh from monkeys and other wild animals found in the developing world. That’s because researchers had identified Patient Zero in the Ebola outbreak as a little boy from Guinea who died in December 2013. Researchers originally believed the toddler had come in contact with the virus through infected bushmeat, probably from a monkey infected by a fruit bat, a suspected reservoir of Ebola (though a new study suggests he may have been infected directly by a bat).

Gire and Yozwiak, using the updates they were getting from Cambridge, were able to demonstrate exactly when and how Ebola had arrived in Sierra Leone: at the funeral of a faith healer who had treated Ebola patients in Guinea. About a dozen people are believed to have become infected during that funeral. That, in turn, triggered the rapid spread around Sierra Leone through human-to-human transmission.

The Broad Institute researchers told government officials it no longer made sense to waste resources warning people about the dangers of eating bushmeat. The lethal vector of Ebola they needed to concentrate on was human-to-human contact.

For Gire, returning to the Sierra Leone lab at the height of the outbreak put his earlier dehydration episode in stark perspective. Back in March, he had nearly collapsed after just four hours in the lab, but now the Kenema workers were each logging 12- to 18-hour shifts, with no days off, racing to get answers.

“Even in the lab, they know that a timely diagnosis can save a patient’s life,” Gire said. “And if it’s negative, getting that news to the hospital quickly can free up a bed for someone else and also save another life.”

But Gire also knew that these exhausted workers were increasing their chances of making a mistake that could prove to be fatal. He tried to identify ways to streamline the lab processes to reduce strain and worker safety risks.

Having worked closely with members of the Kenema lab since 2011, Gire considered them friends as much as collaborators. So, as apprehensive as he was traveling to Sierra Leone as the outbreak was raging, he felt a strong sense of obligation to go himself and help however he could.

He had dreaded one thing even more than making the trip: telling his mother he was going. He knew how much his travel to African hot spots worried her. When he finally called her to break the news, she said in a plaintive whisper, “Oh, Stephen. Please, please be safe.”

In W. Africa, too few doctors

Dolo Nosamiefan degowned on his way out of the Broad Institute lab where samples of the Ebola virus are studied.

Suzanne Kreiter/Globe staff

Dolo Nosamiefan degowned on his way out of the Broad Institute lab where samples of the Ebola virus are studied.

The first recorded outbreak of Ebola happened in Central Africa in 1976, with 318 cases. But the paradox of this virus is that it hasn’t become a global pandemic largely because it is too efficient in its deadliness. Like a greedy worker embezzling so much money from his employer that the company goes bankrupt, Ebola, with a fatality rate north of 70 percent, kills its hosts too quickly to spread as widely as, say, the virus that causes influenza. Previous outbreaks have fizzled out after claiming a few dozen or at most a few hundred lives.

Researchers and public health officials worry the virus will get smarter. It could mutate in ways that allow it to evade diagnosis and treatment. Or it also could acquire the ability to kill less rapidly. Hanging around in the bodies of hosts for longer stretches probably would enable it to infect thousands more.

Although the little Guinean boy was infected a full year ago, his diagnosis wasn’t confirmed until March, by which time Ebola had already spread around Guinea and into neighboring Liberia. Yet Gire pointed out that for those first couple of months after the initial human infection, the virus had spread only to a couple dozen people in Guinea. If public health officials had pounced on the virus during that early period, isolating everyone who had come in contact with it, he said, “they could have caught it while it was still containable.”

Medical resources in West Africa, however, are shockingly sparse. Before the recent outbreak, Sierra Leone, with 6 million people, had just 136 doctors in the entire country, according to the African Health, Human and Social Development Information Service; Ebola deaths have since reduced that number by at least 11. In contrast, Massachusetts, with about the same size population as Sierra Leone, has more than 30,000 active physicians.

The dearth of doctors is even more pronounced in Liberia, where there are only about 50 physicians, according to that same African information service. “It’s essentially zero doctors,” said Yozwiak.

Panic threatens the mission

Despite the speed and breadth of her team’s work, Pardis Sabeti knew it hadn’t been fast enough. She was crushed that Ebola had taken the lives of five of her African coauthors on the Science paper.

Suzanne Kreiter/Globe staff

Despite the speed and breadth of her team’s work, Pardis Sabeti knew it hadn’t been fast enough. She was crushed that Ebola had taken the lives of five of her African coauthors on the Science paper.

In a sleek, glass-walled conference room at the Broad Institute, Pardis Sabeti sat at the head of a blond wood table, for a planning session with a dozen researchers.

Throughout the summer, she had led her team in the hunt to get answers and support their friends in Sierra Leone. In record time, she and Gire and their coauthors had produced a richly detailed paper in the journal Science. It quadrupled the amount of freely available genomic data on Ebola, providing crucial information about the outbreak’s origin and early mutations. Now, Sabeti had convened her team to finalize the schedule and assignments for the sequencing and analysis of the 425 second-wave samples that Gire had brought back with him on the plane, as well as 200 more that followed a short time later. Those samples could reveal how the virus had continued to mutate throughout the summer and into the fall.

The researchers who work for Sabeti speak of the 38-year-old’s brilliant mind and supportive touch, even if they sometimes struggle to keep up with her frenetic approach. “It’s almost as if she’s carrying on three conversations at once with you,” Gire said, “Sometimes, you have to be like: ‘Pardis! What do you need from me right now?’ ”

Sabeti, whose favored form of transportation between her offices at Harvard and at the Broad in Kendall Square is a pair of rollerblades, is forever multitasking. At one point during this pre-Thanksgiving planning session, she indicated her approval for someone’s suggestion by nodding her head vigorously while saying, “Yeah, yeah, yeah!,” then immediately pivoted to mediate a low-level clash between two confident colleagues, all while gathering her wavy hair and rearranging it into a ponytail.

The team’s collection and sequencing of the first wave of samples — 99 in total, harvested from the blood of 78 Ebola patients — during the summer had gone better than anyone could have predicted. Sabeti attributed that to the six years her team had spent working on Lassa fever, with team members Chris Matranga and Kristian Andersen playing critical roles in getting the pipeline ready. Sabeti had urged her team to learn from every setback, such as the time they lost a batch of samples because of a freak car accident during a pit stop in Germany.

Despite the speed and breadth of her team’s work, Sabeti knew it hadn’t been fast enough. She was crushed that Ebola had taken the lives of five of her African coauthors on the Science paper, including her friend Dr. Humarr Khan, the head of the Kenema hospital’s Lassa program.

As an outlet for her grief this summer, Sabeti turned to her other passion: music. The lead singer for the alternative rock band Thousand Days, Sabeti invited a group of visiting scientists from Nigeria to join her in a recording studio. Her team is involved in an effort to train and equip scientists in Nigeria and elsewhere in West Africa so they can eventually do their own genetic sequencing.

The song she wrote for them to record, “One Truth,” was her attempt at capturing her reluctance to accept her colleagues’ deaths and her determination that there be no more.

When Dr. Francis Collins, the director of the National Institutes of Health, was asked to deliver a scientific lecture at the Massachusetts Institute of Technology this fall, he grabbed an acoustic guitar and asked Sabeti to join him on stage to perform the song. He told the crowd the chorus was so simple it would probably stick in their heads for months, with the song’s poignant meaning surely lingering.

Collins later said he hoped that by including Sabeti, “the performance would provide a metaphor for the principle of team-oriented solutions to our world’s greatest problems.” The Ebola fight, with two NIH-supported vaccines heading into Phase II clinical trials, he said, was a perfect demonstration of how “we have to do these things together.”

In a way, it also marked how things had come full circle. In 2011, Sabeti had been invited by Collins to a “Big Idea” conference at NIH, joining leading scientists to present fresh scientific notions.

Sabeti pitched the NIH on an idea she and Gire had developed suggesting the world might be missing major outbreaks of Lassa fever, Ebola, and other emerging infectious diseases simply because many patients didn’t present the most extreme, and recognizable, symptoms.

While the book and movie “The Hot Zone” had fixed in the public imagination the picture of Ebola patients bleeding out of every orifice, Sabeti’s team knew that wasn’t a complete or even particularly representative picture. What if public health officials were missing many Ebola cases in Africa because people were being misdiagnosed as having malaria, or they ended up at hospitals only after excruciating muscle pain had sent them into lethal seizures?

“If we recognize that these viruses are here now, we can head them off before they become a global pandemic,” Sabeti told the group back then. NIH officials were sufficiently impressed to give Sabeti’s team a grant to launch virus surveillance projects in Sierra Leone and Nigeria.

Sitting cross-legged in her office after the pre-Thanksgiving planning session, Sabeti fought back tears when relaying a fresh piece of awful news: The pregnant wife of one of her deceased African collaborators had recently died from Ebola, along with two of their children, leaving the couple’s only surviving child an orphan.

She admitted she was worried about how much information her team would be able to extract from the new samples, which had probably degraded during the months they’d been stored in Sierra Leone under spotty refrigeration.

There was also an extra emotional heaviness to handling this batch, Sabeti said, because it included genetic matter from her deceased African colleagues and coauthors. “So we have this enormous sense of responsibility of what we have to achieve with these samples,” she said.

Sabeti’s team also had to adjust to the new world order that followed the death and drama in Dallas. There, in September, the United States saw its first diagnosed case of Ebola, followed by the news that two of the nurses caring for that patient had also contracted the virus. The widespread panic and increased security measures that ensued created new delays and difficulties that complicated the transfer of serum samples.

Sabeti team’s protocols for rendering the virus inactive and making it ready for transport were now deemed by public safety officials at Harvard and elsewhere to be no longer sufficient. Only after the samples had been treated using the new deactivation agent did someone inform Sabeti, “Oh, by the way, it might kill your samples.”

Things got easier when domestic fears eventually subsided, after the dreaded rush of new American cases never materialized. People came to understand that, for all its lethal power, Ebola is spread far less easily than airborne illnesses. “Unlike with influenza,” Yozwiak said, “almost everyone who gets Ebola knows who they got it from.”

Tracing Ebola’s journey

The Ebola genome is relatively small, only about 19,000 letters, compared with the 3.2 billion letters in the human genome.

Suzanne Kreiter/Globe staff

The Ebola genome is relatively small, only about 19,000 letters, compared with the 3.2 billion letters in the human genome.

Once samples are collected in the field, diagnosed, deactivated, and transported to Cambridge, they go through a four-stage process.

In the “wet lab,” researchers wearing protective gear process the samples. They take the approximately 10 drops of serum in each vial of virus sample and extract its RNA, or nucleic acid. Because Ebola is an RNA virus, that’s where it keeps its genetic code.

When the letters of this code line up in the proper sequence, that becomes the instruction manual the virus uses to copy itself. RNA viruses have a tendency to make errors, or mutations, as they do that copying, since they don’t have the self-correcting capabilities that DNA viruses have.

The Ebola genome is relatively small, only about 19,000 letters, compared with the 3.2 billion letters in the human genome. Still, that code is plenty long to find variations that offer important clues.

To help with the RNA extraction, researchers add ethanol, which, just like when alcohol is mixed with milk, causes curdling. In this case, the curdling is a good thing, because it separates out the nucleic acid.

Normally, this step would be done at the team’s Biosafety Level-2 lab at Harvard. For the second wave of samples, though, Gire and fellow researcher Sarah Winnicki did the processing at Tulane University in New Orleans because Harvard was in the midst of approving the new safety protocols.

The next step involves constructing “libraries” — each containing 96 samples — to go on the sequencing machine.

A few blocks away from the Broad Institute’s main building, in a former beer distribution facility, is the genomics platform. It contains about 50 high-powered sequencing machines,14 of which are the latest and most advanced available.

That allows the Broad to generate data equivalent to about one human genome every minute, according to Andy Hollinger, one of the platform managers. (Sequencing the first human genome took close to a decade.)

Then all that data information is turned over to the team’s computational team, where the final step of analysis begins.

In December, Gire sat around a table with Yozwiak and computational specialist Danny Park. The team had just gone public to the research world with some new results and analysis. As they had suspected, the second-wave samples were proving to be far more degraded than the batch collected early in the summer.

Of the first 96 samples Gire brought back with him from New Orleans, nearly half had turned out to be unusable.

About a quarter offered insufficient results but were deemed worthy of another attempt. Fortunately, though, 21 of the samples proved to be packed with usable information.

Combined with 14 more usable samples sequenced the following week and 10 near the end of December, the team had 45 new sequences, with scores more still to come.

Already, this new collection of 45, representing a random sampling from July through September, told an intriguing and encouraging story: Initial tests gave no indication that the newest mutations would threaten the effectiveness of leading diagnostics and therapeutics in use or in the pipeline.

But in their latest round of analysis they also found a surprise.

In their first sequencing of 99 Ebola genomes this summer, they found two distinct clusters, differentiated by four mutations that collectively served as a sort of watermark. Cluster 1 died out immediately after crossing over from Guinea into Sierra Leone in May, with no secondary transmissions, while Cluster 2 took over. Within two weeks of Ebola’s spread into Sierra Leone, though, there was a new mutation that caused Cluster 2 to branch off.

The team traced this new cluster to a driver who had contracted Ebola after transporting one of the people infected at that fateful funeral of the faith healer. From there, Clusters 2 and 3 continued to spread across Sierra Leone.

In this newest round of samples, the Sabeti team had expected to find both Cluster 2 and Cluster 3 continuing to propagate across Sierra Leona as summer turned into fall. Instead, as Gire stood in front of the conference room whiteboard, he sketched out a map showing their intriguing findings. All indications were that Cluster 2 had died off by early July, and only Cluster 3 was soldiering on.

That could be because lots of Ebola patients from Cluster 3 were “super-spreaders.” While the typical Ebola patient passes the virus on to two people, these super-spreaders infect closer to a dozen.

Yet, this surprising result could have a more worrisome explanation: The mutation at the center of Cluster 3 has some sort of genetic advantage, suggesting the possibility Ebola could be getting smarter on the fly.

As the team rushed to crunch the latest numbers, the news out of Sierra Leone had grown more troubling. The World Health Organization announced that Sierra Leone had surpassed Liberia as having the most Ebola infections. This, too, was surprising. For as limited as Sierra Leone’s medical resources were, they were far more extensive than Liberia’s.

But Sierra Leone, with its operation in Kenema, may have been penalized for its comparative preparedness. The early flood of global aid focused on Liberia and Guinea, largely overlooking Sierra Leone.

Meanwhile, because of its excellent reputation for treating Lassa fever, the Kenema hospital and lab had been overwhelmed with patients, a situation made only more dire after Ebola killed the director of the Lassa program.

Nearby, Nigeria had managed to conquer its outbreak with a rapid containment response. But Nigeria isn’t a fair comparison because its oil revenues have given the country so much more in medical and other infrastructure than its West Africa neighbors. Then again, in outbreaks, infrastructure isn’t always a force for good.

Researchers believe Ebola has been present in very poor parts of Central and West Africa in recent years, but any small outbreaks appear to have fizzled out.

“What kindling had to be set up this time to have this spark ignite?” Yozwiak asked. Then he offered a fascinating theory.

While it’s true the region suffers from alarmingly few medical resources, it has acquired lots of new transportation infrastructure. “In Sierra Leone, you can now drive from one end of the country to the other in four hours on immaculately paved, brand new highways,” Yozwiak said, recalling the journey he and Gire made in July.

Gire cited the relatively new highway that runs from Guinean villages on the border with Liberia and Sierra Leone across the country to the capital city of Conakry.

“You basically see Ebola, as it spreads through Guinea, follows the highway all the way out to Conakry.” The highways, he said, were probably “one of the reasons it was able to spread so quickly.”

Advances in technology and infrastructure can sometimes have unintended consequences, making the world smaller but also enabling a deadly virus to broadcast itself with frightening efficiency. Then again, those same kinds of advances have also helped in the Ebola fight, by closing the distance between West Africa and Cambridge.

Neil Swidey can be reached at swidey@globe.com. Follow him on Twitter @neilswidey.
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