Asking old human tissue to answer new scientific questions

April 16, 2012

Pirates used to say that “dead men tell no tales.” Of course, the buccaneers had never heard of the polymerase chain reaction. Dead men turn out to be loaded with information if you can get your hands on them — or better yet, on small preserved pieces.

The genomics revolution, two decades old, has given biological researchers an astonishing array of tools, both physical and computational, to extract information from once-living tissue. Perhaps the most spectacular example was the discovery of enough remnant DNA in Neanderthal bones to allow scientists to conclude that those extinct hominids once interbred with modern humans.

But it turns out there is a lot of less exotic — and potentially more useful — research to be done on preserved tissue whose age is measured in decades, not millennia.

The best-known examples are the reconstruction of the infamous 1918 Spanish influenza virus from preserved lung tissue, and the discovery of the AIDS virus in blood serum from 1959 and a tissue sample from 1960. Several years ago, scientists used serum collected from airmen during the Korean War to understand the course of hepatitis C infection, a disease unknown at the time the samples were drawn.

This work is likely to increase, for a couple of reasons. Scientists have steadily refined the laboratory technique called polymerase chain reaction (PCR) to catch pieces of DNA swimming in oceans of molecular contamination. Simultaneously, the discovery of new microbes, hormones and biomarkers has given researchers more things to fish for.

The information gained will shed light on why diseases — caused by microbes, environmental exposures and lifestyle changes — emerged when they did. It will reveal how their prevalence has changed over time. It might even help scientists predict future disease trends or outbreaks.

Curiously, nobody has a good idea of how much potentially useful biological material is stored away in universities, hospitals­­, research institutes, museums and private labs. Even the known collections are largely uninventoried and unpublicized.

“I suspect there are incredible collections of things just sitting and waiting for people to take them one step further,” said Edward L. Kaplan, a physician and infectious-diseases researcher at the University of Minnesota Medical School. About 20 years ago, he helped rescue the collection of airmen serum with the help of a refrigerated truck that usually carried frozen pizza.

Reconstructing genomes from degraded fragments is like reconstructing documents that have gone through a shredder. It’s difficult and tedious, and there is no assurance it will work. Most scientists are also reluctant to use up material that sometime in the future might yield its secrets more easily with new tools. There are also privacy issues — and public relations ones — that arise from probing the remnants of dead (or, in some cases, still living) people.

Nevertheless, old tissue sometimes answers questions that nothing else in the world can.

Nine years and still looking

The Spanish flu pandemic of 1918-19 has many mysteries. Where did the virus come from? Why did it kill so many people? Why was it more deadly in people in their 20s and 30s than in people older or younger?

Unlocking these secrets is not an academic exercise. Understanding the pace and pattern of flu evolution — and especially which mutations can change its behavior overnight — is essential for estimating the threat of new strains picked up daily by the global influenza surveillance network.

Jeffery K. Taubenberger of the National Institutes of Health is chipping away at the questions.

Working at the now-defunct Armed Forces Institute of Pathology, he and his collaborators spent nine years reconstructing the virus from three sources. The first fragments were recovered from lung tissue removed at autopsy from American soldiers who died in the epidemic. (The marble-size tissue blocks, chemically fixed so as to not deteriorate and then embedded in wax, have been held at various places in the Washington area since soon after the autopsies.)

Most of the virus’s eight genes, however, were reconstructed from much larger pieces of lung recovered from a body buried in permafrost after flu swept through Brevig Mission, Alaska, killing 90 percent of the Inuit residents. The third source was from flu victims’ tissue blocks stored at Royal London Hospital.

All of those cases came from the epidemic’s explosive “fall wave,” which began in October 1918. There had been scattered fatal cases of flu in Army camps as early as March that year. Was that the same virus? If it was, did it pick up some particularly dangerous mutations over the summer?

Last year, Taubenberger and his team published an analysis of four rare 1918 “spring wave” flu cases, shedding light on what was — and wasn’t — happening in the months before the same virus killed 50 million people worldwide.

The 1918 strain was directly descended from an avian — “bird flu” — virus. Like all flu viruses, it must attach to cells in the nose, throat and lungs before it causes illness. In three of the four spring cases, the gene responsible for that attaching mechanism was more “birdlike” than in the fall cases. By fall, the virus (in most cases) had a mutation that made it better adapted to attacking human beings.

Was that what made the difference in the deadliness of the spring and fall waves? It appears not. The lung damage seen in the spring and fall specimens was nearly identical. There’s also much epidemiological evidence that the spring virus was easily transmitted from person to person, just like the fall one.

So something, still undiscovered, was going on that made the fall wave different. Taubenberger and his research team still hope to find out what.

Their new strategy is to look for strains from the early 1920s, because by then flu was no longer killing young adults at the rate it had a few years earlier. Something had changed. If it turns out there are new mutations in that “Son of Spanish Flu,” then the researchers might be able to deduce the reasons for the parent strain’s lethality.

That’s not the only mystery.

“One of the big questions about the 1918 virus is: Where did it come from?” Taubenberger said. “Having the full [gene] sequence of the virus still hasn’t answered that question,”

But older samples might. They could shed light on the mutations and gene reassortments — small steps and big leaps — that led to the pandemic strain.

Over the past few years, research assistants at the Royal London Hospital have been going through items from the first decade of its pa­thol­ogy collection, which goes back to 1908. They are looking for cases of fatal wintertime pneumonia, illnesses that are most likely to be caused by flu. (However, none of the autopsy reports are labeled ­­“influenza”; that virus wasn’t identified in people until 1933.)

The Royal London team has found 20 candidate cases, and “a handful” have screened positive for flu, Taubenberger said. The work is exceptionally laborious because time has shattered the flu genes into tiny fragments. Just identifying which subtype — there are 16 — these old strains fall into, let alone reconstructing them, will require a lot more testing.

Fishing for the gene fragments requires chopping up pieces of the preserved tissue, extracting its soluble contents with water and then winnowing trace amounts of viral RNA from the human and bacterial RNA that is 99 times more prevalent. The researchers are looking for more efficient ways to do this.

“We could easily burn up all the material just trying to find out what subtype we have,” Taubenberger said. “I have been very reluctant to just dive in. We have waited 110 years, and we can wait a little longer.”

John Oxford, a virologist at Royal London and Taubenberger’s collaborator, agrees.

“Pathologists risked their lives to get those samples,” he says of the tissue from Spanish flu victims. “We need to be able to pass the baton on to another generation. We don’t want to just gobble it up.”

The backstory of HIV

AIDS was identified in the United States in 1981, and the virus that causes it was found in 1983, but nobody credible in the scientific world believes it just appeared overnight.

Human immunodeficiency virus clearly descended from a monkey ancestor, simian immunodeficiency virus. At some point it leaped a “species barrier” and got into people. But the big genetic difference between HIV and SIV implies it must have been evolving for decades — which is to say, circulating in thousands of people — by the time the human disease was recognized.

So what is the unknown backstory of HIV? This is an interesting question of the 20th century that may be answered in the 21st.

“It is amazing to be able to step back in time and look at the genesis of such an important part of human history and human health,” said Michael Worobey, an evolutionary biologist at the University of Arizona.

He is as responsible as anyone for establishing the most credible date for when HIV entered the human population. It occurred between 1884 and 1924, with 1908 the best estimate, based on the genetic differences between SIV and the oldest samples of HIV. Those differences function as a “molecular clock” to measure how much time has passed since the two viruses diverged.

With co-authors from the Democratic Republic of Congo, Belgium and Australia, Worobey published the estimate in the journal Nature in 2008. They used the two oldest known samples of human tissue infected with the AIDS virus — blood plasma drawn in 1959 and a preserved lymph node removed in 1960. Both came from the Belgian Congo (which became the Democratic Republic of the Congo in 1960).

Worobey had heard that there might be human material from the colonial era when he was in Congo in 1990 gathering samples of SIV. After several years and many inquiries, a pathologist at the University of Kinshasa, Jean-Jacques Muyembe, provided him with 27 old tissue samples. Most of these samples did not advance Worobey’s search, but one did: a biopsied lymph node taken from a woman in Leopoldville (the colonial name for Kinshasa). It contained gene fragments of HIV. While that was pay dirt enough, the sample turned out to be unusually informative because it was quite different from the 1959 blood sample, which had been found by another research team.

The 1960 virus was not a direct descendant of 1959’s, but they were both clearly descended from the same SIV that at some point had infected a person — probably a hunter butchering a chimpanzee — and become the founder of HIV.

This “starlike” lineage — multiple viruses recently descended from a single virus — is the signature of an explosive epidemic. It sent a chilling message out of the past: In 1960, more than two decades before the first case of AIDS was reported, HIV was already spreading rapidly through Congo’s population.

“It probably passed over into humans a number of times,” Worobey said. “But something allowed the virus to establish itself this time.”

The timing of when that happened plays a crucial part in two new books.

In “Tinderbox: How the West Sparked the AIDS Epidemic and How the World Can Finally Overcome It,” authors Craig Timberg and Daniel Halperin argue that newly developed trade routes carried HIV out of a jungle fastness in southeastern Cameroon and that prostitution then helped it take hold in Congo’s colonial cities. In “The Origin of AIDS,” French Canadian physician Jacques Pepin cites those facts but also argues that unsterilized needles used in mass treatment campaigns against sleeping sickness, leprosy, syphilis and malaria helped spread the virus widely in the decade or two after SIV entered people.

“If the conclusion had been that the common ancestor of HIV dated back to 1850 or 1800, it would have been difficult to explain,” Pepin said. “But the timing laid out in [Worobey’s] studies fitted perfectly” with the historical events highlighted in the two books.

It may be that a much finer dating of HIV’s emergence will be possible. That’s because it turns out that tissue specimens from about 10,000 Congolese patients that go back to the 1930s still exist, held in Congo, Belgium and Arizona.

The material comes from hospitals in three Congolese cities: Kinshasa, Kisingani (formerly Stanleyville) and Lubumbashi (Elisabethville). The Kinshasa hospital also got biopsy samples from a tuberculosis sanitorium, whose patients 60 years ago were especially likely to have had HIV (as are people with tuberculosis today).

In this yellowing, dessicated, wax-embedded trove there may be hundreds of cases of AIDS waiting to be diagnosed. Given how much can be inferred through genome analysis, the samples might one day tell even more about the AIDS pandemic’s secret childhood.

Next week: Who controls the rare resources of human tissue and sets the rules for their use?

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