This article was originally published by Ebola Deeply.
By Kate Thomas
In this two-part series, Ebola Deeply takes a look at how genomic sequencing is providing insights into the epidemiological and evolutionary course of the Ebola outbreak. We visit Liberia’s genome sequencing center, housed within the Liberia Institute for Biomedical Research (LIBR) and managed by scientists from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID).
The road to Liberia’s genome sequencing center is long and rutted, marked by pools of rainwater dyed orange by the rust-colored soil. A quiet hero in the battle against Ebola, the center sits on the campus of LIBR, where groundbreaking hepatitis research was once conducted on chimpanzees. Funded by the U.S. Department of Defense and partnering with the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), the center is run by USAMRIID and operates in conjunction with an Ebola diagnostics lab.
Inside the Genome Sequencing Lab
Lawrence Fakoli is Liberia’s first genetic sequencer. Dressed in a lab coat and a pair of lilac medical gloves, he uses a long pipette to transfer a sample into a centrifuge tube. By his side is an Illumina MiSeq machine — a supercomputer capable of crunching genetic code — and a bottle of DNAZap, a reagent that eliminates possible contaminating DNA on contact.
Fakoli, who trained in Ghana and Egypt, has worked at the Liberia Institute of Biomedical Research for five years.
“I was the first Liberian to be trained in Ebola diagnostics,” Fakoli says, leaning forward as he talks. “After I was comfortable with that, I started genome sequencing. I knew nothing about it to start with, but because of the Ebola outbreak, I’ve become interested in it. I’m encouraged by how important this field could be for Liberia. But the country is like a virgin when it comes to genetic research; nobody knows much about it.”
Genome sequencing enables researchers to track the way a virus mutates over time. RNA viruses such as Ebola have high mutation rates; analyzing the genome is like turning the pages of the virus’s personal diary. The Ebola virus has mutated hundreds of times since the beginning of the outbreak, and although none of these mutations are believed to have changed the characteristics of the virus, they reveal critical information. Sequencers, such as a team led by Harvard geneticist Pardis Sabeti, have been able to sketch the arc of the Ebola outbreak, tracking its movements through an area, country or region.
“We’re still finding out how whole genome sequencing fits into outbreak response, such as with Ebola virus disease,” says Michael Wiley, a virologist contracted to USAMRIID, currently working at LIBR with Fakoli for a few weeks. “It is still a novel concept to get whole genomes in a short amount of time, allowing us to piece together the full picture of an outbreak — while it’s still ongoing.”
Wiley and Fakoli are currently doing just that; using Illumina sequencing technology to glue together clues in Liberia’s new Ebola resurgence. On June 29, 17-year-old Abraham Memaigar — who lived in the same area as the center — died, and a routine oral swab from his body tested positive for Ebola. Within days of Memaigar’s death, Wiley was able to determine that the viral strain in the boy’s body was genetically similar to that circulating in this part of Liberia last year. He ruled out cross-border transmission from Sierra Leone or Guinea. Further sequencing has revealed that the five additional cases diagnosed since, including one death, are part of the same Liberian viral cluster.
“In this new pocket of infection, the virus looks as it did in Liberia last July or August,” Wiley said. “The virus hasn’t acquired the same amount of mutations as expected. The analogy would be taking a frozen vial from a year ago, and sequencing it.”
Health authorities in Liberia still don’t know how the patients in the new cluster became infected. But because Wiley and his team could match the new viral infections to an earlier Liberian strain, they have been able to dismiss some possibilities. A theory involving a dead dog, cooked and eaten by Memaigar and at least two other patients, are among them. The team says it is unlikely that the virus sprang from a reservoir, or carrier host, such as a dog.
“If it was a reservoir case, the virus would still be replicating in the reservoir, so you’d expect to see changes in the virus over time,” Wiley said. “It seems most likely that these new cases are coming from a survivor. We have seen this before from viral RNA extracted from samples coming from an immune-privileged site, like semen from a survivor. The virus possibly replicates at a slower rate acquiring less mutation overtime then expected when compared to human-to-human transmission. But we are looking into all the possibilities to explain this strange event.”
Positives and Negatives
Brian Kearney, currently the mission’s lead diagnostic scientist, didn’t expect his career to include testing the bones of a dead dog for Ebola. He flew back to Liberia four days before the new cluster of cases emerged, expecting to lead ongoing Ebola diagnostic work and build capacity among local staff.
In his role for USAMRIID, Kearney works on diagnostic assays — investigative procedures that quantitatively measure pathogen levels in a cell or organism. He and his team came to Liberia at the height of the outbreak last year, providing Ebola diagnostic support to the country’s weak, overwhelmed health system. They set up a scientific training center where Liberian scientists were taught how to use assays, while routine biosurveillance for Ebola continued. From March 9 to June 29, every sample received by the lab tested negative.
“Then the new cluster started,” Kearney says. “I went into the lab as usual and did assays on the oral swab of the 17-year-old who died. I did my assays three times, I rextracted three times. They came out positive. The next day, the Centers for Disease Control (CDC) exhumed the body and took some blood from the heart. The result was the same.”
“With sequencing, just one test could reveal much more than malaria or typhoid.””
Megan Wisniewski, the operations officer in charge of USAMRIID’s mission, says the positive result was a shock.
“That test was a definite surprise because we had been Ebola-free for so long, but it was definitely not out of the realm of possibility,” Wisniewski says. “That’s why it’s still important to continue surveillance to catch cases like this. The CDC and WHO are the big pushers behind what needs to be done as far as biosurveillance and as an international partner we’re here to support that. My understanding is that they recommend 90 days of surveillance after the 42-day countdown to Ebola-free status.”
As health officials raced to understand the source of the new cluster, they managed to find the remains of a dog that Memaigar’s relatives believed he ate before falling sick. Along with the bones of one of the dog’s puppies, the skeleton was brought to Kearney.
“It was basically a skin and bones experiment,” Kearney says. “Nobody had ever taken a dog bone for an extraction, until now. I took some of the tissue from the fur and minced it up as finely as I could. I thought if the Ebola virus was in there, I might be able to extract some of its proteins. If there was viral RNA in there, I theoretically would have been able to see it. I took a needle and flushed a solution through the bones and did an extraction. Everything was negative.”
“If there was viral RNA in the bones, I theoretically would have been able to see it”
Although Kearney didn’t have much to work with, the test used by USAMRIID is highly sensitive. It can detect very low levels of viral RNA, even once the virus has died — evidence enough to discredit the theory that the dog could have served as a reservoir for the virus.
“If viral strands were in the skin or bones and weren’t too damaged, we could have detected them,” Kearney says. “The virus can get killed but some viral strands would probably remain. If the dog were a reservoir, wouldn’t we see the virus keep coming back? How could this dog do something that no other dog could do?”
He says the dog story reminds him of the Beltway sniper attacks that rocked Washington D.C., Maryland and Virginia in 2002.
“Witnesses remembered seeing a white panel van,” Kearney says. Everybody was stuck on that white panel van. When they finally found the shooter, he had an old sedan car. People focused on one event so much that they missed other clues. There’s something else out there.”
The Hot Zone
Clues as to what that something else might be will likely be brought here, to the hot zone. Every day, the center receives dozens of oral swab tests and whole blood samples from all over the country, as part of ongoing measures to ensure biosurveillance for Ebola.
James Kolbah, a lab aid who assists technicians with diagnostics, is walking past. He carries a pair of green flasks containing new samples, which he leaves in an enclosed, safe area on the edge of the hot zone. Closing the door to the safe area, he steps back and watches as a technician clad in personal protective equipment comes forward to collect them.
“Today we’ve had 19 samples so far,” Kolbah says. “During the worst of Ebola, we received about 175 every day. I love my lab job, even though it can feel dangerous. I thank God that none of my family came down with Ebola because of me. If I had my own powers like God, I would stop Ebola just like that. But I’ve got different powers instead, to receive the samples and bring them. I can do this work.”
According to Wisniewski, there are plans to continue local capacity building and training in biosurveillance and diagnostics long after the current cluster of cases is over.”
“We have four trained Liberian researchers and one in training,” Wisniewski says. “They’re the ones who come in and perform the Ebola testing. We’re also fortunate to have lab aids for each day. They’re really dedicated to the cause. I understand that probably everyone in Liberia has been affected by this outbreak. People have lost family members, people have lost friends, so seeing their dedication to continue the mission is inspirational. It’s great to be here and to be able to mentor and assist in the field.”
“There’s a number of endemic diseases here that are important to West Africa and also globally, so instead of trying to come here and collect samples and just take them back to the U.S., this is an opportunity to come here and be able to train the local staff to do the science themselves,” Wisniewski adds.
Next Generation Sequencing
Back in the genome sequencing laboratory, the screen of the MiSeq Illumina machine lights up. It is the older cousin of the HiSeq X Series, a piece of machinery that revolutionized genomic technology when it was launched last year. It birthed the $1000 genome, cutting the price of full genome sequencing by ten. With the MiSeq, probes, reagents and magnetic beads crunch superfluous DNA, leaving a sample of pure Ebola virus, plus the necessary controls.
Wiley and Fakoli are now breaking down lineages, analyzing sequencing data from two more cases in Liberia’s Ebola resurgence. They want to understand another mystery; why the viral load from these two samples is weak. Cycle thresholds of polymerase chain reaction are used to determine viral load; anything in the high 30s is generally deemed low.
“When the second and third cases were found, it seemed they were already in the late stages of infection,” Wiley says. “But their symptoms were mild, and their cycle thresholds were in the high 30s, pushing 40.”
The death of 17-year-old Memaigar may have alerted responders to the new cluster, but Liberian health authorities say it’s possible that he wasn’t the index case. Although the other patients were diagnosed later, one of them might have contracted Ebola first, spreading it to Memaigar. If their own antibody response was sufficient to tackle the virus without treatment, it could explain why their symptoms and viral loads were more subtle than expected. That could mean that other cases might be going undetected in Liberia.
Further sequencing will help determine the differences in viral mutations between the patients, allowing the team to piece together the timeline of the latest cluster.
“We’re also pulling some more samples from that time period, to see if there’s anything we haven’t noticed before,” Wiley adds. “I personally think it would be interesting to sequence all the samples they have here, to get a really good picture of the whole outbreak. Right now we have just 1 percent of the cases sequenced, but if we were able to sequence even just 10 percent, we might be able to determine whether these kinds of strange clusters have also happened elsewhere.”
At the moment, the laboratory is only running filovirus panels, but technology is rapidly boosting the capacity of genomic surveillance. Next generation sequencing could eventually become a diagnostic tool. And if it came to Liberia, it could change the landscape of healthcare.
“With sequencing, you can potentially test for as many possible pathogens as you can find,” Fakoli says. “You could go to the hospital and just get one test, and they’d tell you what illness you have. At the moment, most Liberian clinics only have the capacity to test for malaria or typhoid. With sequencing, just one test could reveal much more than malaria or typhoid.”
Fakoli says he hopes to train other Liberian scientists in genome sequencing.
“I attended a conference on genome analysis in the U.S. in May,” he says. “This year was the 10th year it has run, but I’m the first Liberian to ever attend. Other African countries had two or three people there. They would say to me, “Oh, you’re from Liberia? Where are your colleagues?” And I would look around. It was only me. “It would mean a lot to have more Liberians trained and working in genome sequencing.”
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Kate Thomas is a contributor to Ebola Deeply.
[Photo courtesy of Ebola Deeply]