This is the context that brought DRC60 out of Kinshasa. A senior Congolese virologist named J. J. Muyembe, aware of the archived pathology specimens at the University of Kinshasa and equally aware of the OPV debate, took it upon himself to enlarge the body of available data. Muyembe went up to the university, rifled through the pantry behind the blue curtain, packed 813 paraffin-embedded specimens into an ordinary suitcase, and carried it with him on his next professional visit to Belgium. There he handed the trove to a colleague named Dirk Teuwen, who had taken part in the Royal Society meeting a couple years earlier. Teuwen, in accord with a prior agreement for collaborative study, sent them to Michael Worobey in Tucson.
These two lines of narrative fold back into each other. Worobey, as a grad student, knew both Bill Hamilton at Oxford and some of the disease biologists in Belgium. Impelled by his own interest in the origins of HIV, Worobey accompanied Hamilton to Congo on that last fatal fieldtrip. They went in January 2000, during the chaotic aftermath of a civil war, which had replaced the longtime potentate Mobutu Sese Seko with the upstart Laurent Kabila as president of the DRC. Hamilton wanted to collect fecal and urine samples from wild chimpanzees; those specimens, he hoped, might help confirm or refute the OPV theory. Worobey, for his part, put little stock in the OPV theory but wanted more data from which to chart the origin and evolution of HIV. It was a crazy time in the Democratic Republic of the Congo, more crazy than usual, because two rebel armies opposed to Laurent Kabila still controlled much of the eastern half of the country. Hamilton and Worobey flew into Kisangani (formerly Stanleyville), a regional capital along the upper Congo River, the same city where Koprowski had begun his Congo enterprise. Now it was occupied by Rwanda-backed forces on one riverbank and Uganda-backed forces on the other. Commercial airlines weren’t flying, because of the war, so the two biologists shared a small, chartered plane with a diamond dealer. In Kisangani they paid their respects to the Rwanda-backed commander, whose ambit included most of the city, and as quickly as possible got out into the forest, where they would be safer among the leopards and snakes. They spent a month collecting fecal and urine samples from wild chimpanzees, with help from local guides, and by the time they left, Hamilton was sick.
Neither he nor Worobey knew how sick, but they caught the next exit flight they could, which took them to Rwanda. From there they bounced to Entebbe in Uganda, where Hamilton got a confirmed diagnosis of falciparum malaria and some treatment, then onward to Nairobi, and from Nairobi up to London Heathrow. By now Hamilton seemed past the worst of his illness; he was feeling much better. They had accomplished their mission and life was good. An American field biologist once expressed to me how he felt in such moments. “That’s the name of the game: getting home with the data.” This man’s research too involved dangers—shipwreck, starvation, drowning, snakebite, though not malaria and Kalashnikov rifles. “If you take too many risks, you don’t get home,” he said. “If you take too few, you don’t get the data.” Hamilton and Worobey got the data, got home, then learned that the ice cooler containing their precious chimp specimens had gone astray in luggage handling somewhere between Nairobi and London.
I visited Michael Worobey in Tucson to hear about all this. “Everything was fine,” he told me, “except we checked six bags, including the cooler that had samples, and five of our bags came through the carousel and the one with the samples disappeared.” His friend Hamilton, feeling ill again the next morning, went to a hospital—and hemorrhaged disastrously, perhaps due to anti-inflammatory drugs he’d been taking against the malarial fever. Worobey phoned and got the news from Hamilton’s sister: Who are you why are you calling Bill is in extremis. Worobey meanwhile had been hassling by long-distance phone with a luggage handler in Nairobi, who assured him that the cooler had been found and would arrive on the next flight. What arrived was someone else’s cooler—full of sandwiches or somesuch, as he recalled. “So that was an extra bit of drama that unfolded as Bill was dying in the hospital,” Worobey told me. The correct cooler arrived two days later but Hamilton was in no shape to celebrate. He went through a series of surgeries and transfusions and then, after weeks of struggle, he died.
The fecal samples from Congolese chimps, for which Hamilton had given his life, yielded no SIV-positives. A couple of urine samples registered in the borderline zone for antibodies. Those results weren’t clear or dramatic enough to merit publication. Good data are where you find them, not always where you look. Several years later, when the human pathology samples from Kinshasa reached Tucson—those 813 little blocks of tissue in paraffin, the ones J. J. Muyembe had carried to Belgium in a suitcase—Michael Worobey was ready. He found DRC60 among them, and it told an unexpected story.
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Screening paraffin-embedded hunks of old organ samples to find viral RNA isn’t easy, not even for an expert. Those little blocks, Worobey said, turned out to be “some of the nastiest kinds of tissues to do molecular biology with.” The problem wasn’t forty-three years at room temperature in a dusty equatorial pantry. The problem was the chemicals used in fixing the tissues—the 1960 equivalent of the beakers of methanol and xylol that Professor Kabongo had shown me. Back in those days, pathologists favored something called Bouin’s fixative, a potent little mixture containing mostly formalin and picric acid. It worked well for preserving the cellular structure of tissues, like salmon in aspic, so that samples could be sliced thin and examined under a microscope; but it was hell on the long molecules of life. It tended to break up DNA and RNA into tiny fragments, Worobey explained, and form new chemical bonds, leaving “sort of a big, tangled mess rather than a nice string of beads that you can do molecular biology on.” Because the process was so laborious, he screened just 27 of the 813 tissue blocks from Kinshasa. Among those twenty-seven, he found one containing RNA fragments that unmistakably signaled HIV-1. Worobey persisted adeptly, untangling the mess and fitting the fragments to assemble the sequence of nucleotide bases he named DRC60.
That was the wet work. The dry work, done largely by computer, entailed base-by-base comparisons between DRC60 and ZR59. It also involved broader comparisons, placing those two within a family tree of known sequences of HIV-1 group M. The point of such comparisons was to see how much evolutionary divergence had occurred. How far apart had these strains of virus grown? Evolutionary divergence accumulates by mutation at the base-by-base level (other ways too, but those aren’t relevant here), and among RNA viruses such as HIV, the mutation rate is relatively fast. Equally important, the average rate of HIV-1 mutation is known—or anyway, it can be carefully estimated from the study of many strains. That rate of mutation is considered the “molecular clock” for the virus. Every virus has its own rate, and therefore its own clock measuring the ticktock of change. The amount of difference between two viral strains can therefore reveal how much time has passed since they diverged from a common ancestor. Degree of difference factored against clock equals elapsed time. This is how molecular biologists calculate an important parameter they call TMRCA: time to most recent common ancestor.
Okay so far? You’re doing great. Take a breath. Now those bits of understanding will boost us across a deep gulf of molecular arcana to an important scientific insight. Here we go.
Michael Worobey found that DRC60 and ZR59, sampled from people in Kinshasa during almost the same year, were very different. They both fell within the range of what was unmistakably HIV-1 group M; neither could be confused with group N or group O, nor with the chimp virus, SIVcpz. But within M, they had diverged far. How far? Well, one section of genome differed by 12 percent between the two versions. And how different was that, measured in time? About fifty years’ worth, Worobey figured. More precisely, he placed the most recent common ancestor of DRC60 and ZR59 in the year 1908, give or take a margin of error.
Spillover back in 1908? That’s much earlier than anyone suspected, and therefore the sort of discovery that gets into an august journal such as Nature. Publishing in 2008, a century after the
fact, with a list of coauthors that included J. J. Muyembe and Jean-Marie Kabongo, Worobey wrote:
Our estimation of divergence times, with an evolutionary timescale spanning several decades, together with the extensive genetic distance between DRC60 and ZR59 indicate that these viruses evolved from a common ancestor circulating in the African population near the beginning of the twentieth century.
To me he said: “This wasn’t a new virus in humans.”
Worobey’s work directly refuted the OPV hypothesis. If HIV-1 existed in humans as early as 1908, then obviously it hadn’t been introduced via vaccine trials beginning in 1958. Clarity on that point was valuable—but only a side benefit of Worobey’s contribution. Even more important was the basic fact of his early date. Placing the crucial spillover in time, so long ago, represented a big step toward understanding how the AIDS pandemic may have started and grown.
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Placing the spillover in space was equally important, and achieved by a different laboratory. Beatrice Hahn is somewhat older than Worobey and had begun her work on the origin of AIDS long before he found DRC60.
Born in Germany, Hahn took a medical degree in Munich, then came to the United States in 1982 and spent three years as a postdoc in Robert Gallo’s lab, studying retroviruses. She moved next to the University of Alabama at Birmingham, where she became Professor of Medicine and Microbiology and codirector of a center for AIDS research, with a group of bright postdocs and grad students working under her aegis. (She remained at Alabama from 1985 to 2011, a period encompassing most of the work described here, and then joined the Perelman School of Medicine at the University of Pennsylvania, in Philadelphia.) The broader purpose of Hahn’s various projects, and the goal she shares with Worobey, is to understand the evolutionary history of HIV and its relatives and antecedents. The fittest label for that sort of research is the one Worobey mentioned when I asked him to describe his field: molecular phylogenetics. A molecular phylogeneticist scrutinizes the nucleotide sequences in the DNA or RNA of different organisms, comparing and contrasting, for the same reason a paleontologist scrutinizes fragments of petrified bone from extinct giant saurians—to learn the shape of lineages and the story of evolutionary descent. But for Beatrice Hahn especially, as a medical doctor, there’s an additional purpose: to detect how the genes of HIV function in causing disease, toward the prospects of better treatment, prevention, and maybe even a cure.
Some very interesting papers have come out of Hahn’s laboratory in the past two decades, many of them published with a junior researcher as first author and Hahn in the lab leader’s position, last. That was the case in 1999, when Feng Gao produced a phylogenetic study of SIVcpz and its relationship to HIV-1. At the time there were only three known strains of SIVcpz, all drawn from captive chimps, with Gao’s paper adding a fourth. The work appeared in Nature, highlighted by a commentary calling it “the most persuasive evidence yet that HIV-1 came to humans from the chimpanzee, Pan troglodytes.” In fact, Gao and his colleagues did more than trace HIV-1 to the chimp; their analysis of viral strains linked it to individuals of a particular subspecies known as the central chimpanzee, Pan troglodytes troglodytes, whose SIV had spilled over to become HIV-1 group M. That subspecies lives only in western Central Africa, north of the Congo River and west of the Oubangui. So the Gao study effectively identified both the reservoir host and also the geographical area from which AIDS must have arisen. It was a huge discovery, as reflected in the headline of Nature’s commentary: FROM PAN TO PANDEMIC. Feng Gao at the time was a postdoc in Hahn’s lab.
But because Gao based his genetic comparisons (as Martine Peeters had done earlier) on viruses drawn from captive chimps, the soupçon of uncertainty about infection among wild chimpanzees remained, at least for a few more years. Then, in 2002, Mario L. Santiago topped a list of coauthors announcing in Science their discovery of SIVcpz in the wild. Santiago was a PhD student of Beatrice Hahn’s.
The most significant aspect of Santiago’s work, for which he got his richly deserved doctorate, was that on the way toward detecting SIV in a single wild chimpanzee (just one animal among fifty-eight tested), he invented methods by which such detections could be made. The methods were “noninvasive,” meaning that a researcher didn’t need to capture a chimp and draw its blood. The researcher needed only to follow animals through the forest, get under them when they pissed (or, better still, send a field assistant into that yellow shower), collect samples in little tubes, and then screen the samples for antibodies. Turns out that urine could be almost as telling as blood.
“That was a breakthrough,” Hahn told me, during a talk at her lab in Birmingham. “We weren’t sure it would work.” But Santiago took the risk, cooked up the techniques, and it did work. The very first sample of SIV-positive urine from a wild chimpanzee came from the world’s most famous community of chimps: the ones at Gombe National Park, in Tanzania, where Jane Goodall had done her historic field study, beginning back in 1960. That trace of virus didn’t match quite as closely with HIV-1 as Feng Gao’s had done, and it came from a chimp of a different subspecies, the eastern chimpanzee, Pan troglodytes schweinfurthii. But it was SIVcpz nonetheless.
The advantage of sampling at Gombe, Hahn told me, was that those chimps didn’t run away. They were truly wild but, after four decades of study by Goodall and her successors, well habituated to human presence. For use elsewhere, the urine-screening method wasn’t practical. “Because, you know, non-habituated chimps don’t stay close enough so you can catch their pee.” You could collect their poop from the forest floor, of course, but fecal samples were useless unless preserved somehow; fresh feces contain an abundance of proteases, digestive enzymes, which would destroy the evidence of viral presence long before you got to your laboratory. These are the constraints within which a molecular biologist studying wild animals labors: the relative availability and other parameters of blood, shit, and piss.
Another of Hahn’s young wizards, Brandon F. Keele, soon solved the problem of fecal sample decay. He did it by tinkering with a liquid stabilizer called RNAlater, a commercial product made by a company in Austin, Texas, for preserving nucleic acids in tissue samples. The nice thing about RNAlater is that its name is so literally descriptive: The stuff allows you to retrieve RNA from a sample . . . later. If it worked with RNA in tissues, Keele reasoned, maybe it could work also with antibodies in feces. And indeed it did, after he and his colleagues untangled the chemical complications of getting those antibodies released from the fixative. This technique vastly enlarged the scope of screening that was possible on wild chimpanzees. Field assistants could collect hundreds of fecal samples, scooping each into a little tube of RNAlater, and those samples—stored without refrigeration, transported to a distant laboratory—would yield their secrets later. “If we find the antibodies, we know that chimps are infected,” Hahn told me. “And then we can home in on those we know are infected, and try to get the viruses out.” Antibody screening is easy and quick. Performing PCR amplification and the other requisite steps to probe for fragments of viral RNA is far more laborious. The new methods allowed Hahn and her group to look first at a large number of specimens and then work more concertedly on a select few. They could separate the Shinola from the shit.
And they could expand their field surveying beyond Gombe. They could turn their attention back to Pan troglodytes troglodytes, the subspecies of chimp whose SIVcpz most closely matched HIV-1. Working now with Martine Peeters of Montpellier, plus some contacts in Africa, they collected 446 samples of chimpanzee dung from various forest sites in the south and southeast of Cameroon, after which Brandon Keele led the laboratory analysis. DNA testing showed that almost all the samples came from P. t. troglodytes (though a couple dozen derived from a different chimp subspecies, P. t. vellerosus, whose range lay just north of a major river). Keele then looked for evidence of virus. The samples yielded two surprising results.
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To hear about those surprises, I visited Brandon
Keele, who by this time had finished his postdoc with Hahn and gone off to a research position at a branch of the National Cancer Institute, in Frederick, Maryland. He was still studying viral phylogenetics and AIDS, as head of a unit devoted to viral evolution. His new office and lab were on the grounds of Fort Detrick, a high-security installation that once housed the U.S. biological weapons program and still encompasses USAMRIID, the big army research institute on infectious diseases. Since I was entering without an escort, soldiers at the guardhouse searched the underside of my rental car for a bomb before letting me pass. Keele, waiting to flag me down outside the door of his building, wore a blue dress shirt, jeans, his black hair moussed back, and a two-day stubble. He is a tall young man, extremely polite, raised and educated in Utah. We sat in his small office and looked at a map of Cameroon.
The first surprise to emerge from the fecal samples was high prevalence of SIVcpz in some communities of Cameroonian chimps. Two that scored highest, Keele said, were at sites labeled Mambele (near a crossroads by that name) and Lobeke (within a national park). Whereas all other sampling of chimps had suggested that SIV infection was rare, the sampling in southeastern Cameroon showed prevalence rates up to 35 percent. But even there, the prevalence was “spotty,” Keele said. “We can sample hundreds of chimps at a site and find nothing.” But go just a little farther east, cross a certain river, sample again, and the prevalence spikes upward. That was unexpected. The rates were especially high in the farthest southeastern corner of the country, where two rivers converge, forming a wedge-shaped national boundary. This wedge of Cameroon appears to jab down into the Republic of the Congo (not to be confused with the DRC), its neighbor to the southeast. The wedge was a hotspot for SIVcpz.
The Chimp and the River: How AIDS Emerged from an African Forest Page 5