Even a scrap of old DNA can yield vital clues about the history of a disease. So when Oliver Pybus, an evolutionary biologist at Oxford University, heard about the short sequence of viral genome extracted from blood kept frozen for more than 60 years, he had to have it. With that genetic sliver, Pybus thought he could add a vital chapter to a story he has long sought to complete: the history of the hepatitis C virus.
Scientists have amassed a wealth of knowledge about hepatitis C since its discovery in 1989. The virus currently infects about 150 million people worldwide and exists in several variations, or genotypes. Roughly 3 million people are infected in the U.S., mostly as a result of blood transfusions before the mid-1970s, when paid blood donations were stopped. (People infected with hepatitis C, often through contaminated needles from injection drug use, accounted for a large swath of paid donors.)
The hepatitis C virus can remain silent for decades as it colonizes the liver and may never harm its human host. But in about a third of those infected, hepatitis C can cause cirrhosis, liver cancer and death if untreated.
After years of difficult and inadequate treatments, two drugs — Harvoni and Viekira Pak, approved in 2014 — eradicate the virus in more than 90 percent of patients (though their high cost — around $80,000 for a 12-week course of treatment — has come under continual fire).
Still, mysteries remain. No one knows the natural course of the disease over a human lifetime, or how the virus spread around the world in the first place. The virus mutates exceptionally fast — “a million times faster than our own genome,” says Pybus.
Pybus studies the evolution of viruses using genetic sequences and computer software. Biologists have used mathematics to infer viral ancestry — turning back the molecular clock, they call it — since the 1960s. Based on coalescent theory, an approach to population genetics that uses genetic diversity to trace genealogies, Pybus developed calculations that generate approximate chronologies of viral epidemics. Equipped with nothing more than genomes and the rate of mutation — no blood samples or medical histories required — Pybus has uncovered the history of HIV, various flu viruses and other pathogens. His methods earned him some fame with the Centers for Disease Control and Prevention when he and others found the origin of the 2009 swine flu outbreak.
Like a viral cartographer, Pybus has also mapped the journey of hepatitis C from Ghana to the Caribbean along trans-Atlantic slave trade routes between 1700 and 1850, and from the Netherlands to Indonesia and Surinam during Dutch colonial rule. His calculations revealed the movement of hepatitis C from Central Africa to Egypt sometime between 1860 and 1925. Pybus theorized that Tunisian troops carried the virus home from the Democratic Republic of Congo after the United Nations stationed them there in the 1960s during the Congo crisis.
But the map is sketchy, owing to the lack of older evidence. The more recent the sequence, the less precise the history. Only older genomes could make the map more exact. And now Pybus had his most powerful tool yet: the oldest genetic evidence of hepatitis C.
How such evidence became available begins with another infectious agent: Streptococcus pyogenes, the bacterium that causes strep throat. In the mid-1940s, the Department of Defense commissioned researchers, led by infectious disease scientist Charles Rammelkamp, to study whether treating strep with penicillin would also prevent rheumatic heart disease, then a serious problem among troops. The team focused on Warren Air Force Base in Wyoming, where strep was common, collecting some 45,000 blood samples from more than 9,000 recruits between 1948 and 1954.
After they got their answer — yes, penicillin could prevent rheumatic heart disease — Rammelkamp, recognizing the samples’ potential value to future research, kept the vials in basement freezers at Cleveland’s Case Western Reserve University, where he held a professorship until retiring in the mid-1970s.
Rammelkamp’s colleague, Edward Kaplan, a pediatric infectious disease specialist and pediatric cardiologist at the University of Minnesota Medical School, then took the baton. “It’s a priceless collection,” he says, but its future was precarious; Kaplan knew the samples would be discarded if he didn’t act to preserve them. And act he did: The resourceful Kaplan got a local trucking concern to transport the vials from Cleveland to Minneapolis in a frozen pizza truck for free, provided they could tout the contribution to medical research in their union magazine.
In the mid-1990s, Leonard Seeff, a hepatologist at the Veterans Administration Hospital in Washington, D.C., overheard Kaplan discussing the Warren Sera Collection, as it came to be known. Seeff immediately wondered whether any of the blood samples contained antibodies to the hepatitis C virus. If they did, Seeff could answer a question that had been impossible to investigate: How prevalent was hepatitis C in the 1940s and ’50s before it was even known to exist? No one knew. But if the virus was present in these frozen samples, Seeff could study the 40-year course of the disease in an instant.
“He said if you find any positives for hepatitis C, I’ll give you a bottle of champagne,” Kaplan recalls.
The samples were defrosted for the first time in 40 years, and more than 8,500 army recruits were retroactively tested for the virus. Seventeen were positive. Hepatitis C had been at the Warren Air Force Base in the 1950s. And eight of those recruits were still alive.
Zelma Buskell, Seeff’s study coordinator, traveled across the country to meet most of the veterans and take new samples. Not any of them knew how they got the virus, though one veteran remembered injecting drugs during that time, to Buskell’s surprise.
“You think of drug use as starting with Vietnam,” she says. Finding the recruits in generally good health contradicted the grim outlook most physicians held for hepatitis C patients. “People who’d been infected 40 years earlier were still alive,” says Seeff, who reported his results in 2000. “It was not inevitably a fatal disease.”
A First Date
The story could have ended there, except for one inquisitive scientist at the National Institutes of Health. In 2001, Yasuhito Tanaka, now at Nagoya University, spent a year in the NIH laboratory of Harvey Alter, the virologist largely credited with the discovery of hepatitis C. Tanaka wanted to know when hepatitis C entered the U.S., so he asked Seeff for viral samples to sequence.
After making his way through just 4 percent of the viral genome, Tanaka found he couldn’t complete the sequences and set the project aside. It was all but forgotten until Pybus heard about it in 2011 from another Japanese colleague and asked Tanaka to send what he had.
Pybus suspected his methods, unavailable just a few years earlier, could wring some vital history out of Tanaka’s work. Tanaka had sequenced the middle region of the code for NS5B, the enzyme that makes new genome copies during replication. Pybus compared the sequences with genomes of modern-day hepatitis C virus. The number of differences between them, explains Pybus, “is indicative of how long it’s been since they shared a common ancestor.”
The study confirmed that the 1953 strain was genotype 1B, one of the epidemic subtypes of the virus and a common strain worldwide. Pybus then calculated when the sequences would have last been alike: in 1901, give or take about 25 years. “All subtype 1B infections today are descended from that one 1B infection back at the turn of the century,” he says.
Pybus speculates that the virus first arrived in the U.S. decades earlier in the blood of West Africans sold into slavery. This route could explain why African-Americans were historically less responsive to some treatments for hepatitis C: The virus may have “increased its ability to infect and persist in individuals with that genetic background,” he notes. Sequencing the rest of the 1953 genome samples could yield further insights, and Pybus also hopes to find more such ancient treasures, which help shed light on our genetic relationship with diseases.
As for Kaplan, he’s still waiting for his bottle of champagne from Seeff. The blood samples he kept safe for so long are waiting, too. In 2009, Kaplan transferred the samples back to the care of the government. They currently sit frozen at Wright Patterson Air Force Base in Ohio, ready for the next researcher to defrost a mystery.