The Chimp and the River: How AIDS Emerged from an African Forest (7 page)

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 SIV
_cpz
and its relationship to HIV-1. At the time there were only three known strains of SIV
_cpz
, 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 SIV
_cpz
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 SIV
_cpz
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 SIV
_cpz
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.

11

T
o
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 SIV
_cpz
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 SIV
_cpz
.

The second surprise came once he extracted viral fragments from the samples, amplified those fragments, sequenced them, and fed the genetic sequences into a program that would compare these new strains with many other known strains of SIV and HIV. The program expressed its comparisons in the form of a most-probable phylogeny—a family tree. Keele recalled watching the results for a certain chimp, an individual labeled LB7, whose feces had been collected at Lobeke. “We were just shocked,” he said. “I mean, I had ten people around my computer, all waiting to see what that sequence looked like.” What it looked like was the AIDS virus.

When his computer delivered its latest tree, LB7’s isolate of SIV
_cpz
showed up as a twig amid the same little branch that held all known human strains of HIV-1 group M. (In scientific lingo, it fell within the same
clade.
) It was at that point “the closest thing” to a match, Keele told me, that had ever been found in a wild chimp. “And then we find more, right? The more we dig, the more we find.” The other close matches came from that same little area: southeastern Cameroon. A chilling, historic epiphany, at which Keele and his colleagues were thrilled. “You can’t make this stuff up, as Beatrice would say. It’s too good.” Their joy lasted about ten seconds, after which everyone became hungry for more samples and more results. Your celebration is always provisional, Keele told me, until you’ve written the paper and gotten that congratulatory note of acceptance from the editors of
Science.

Keele and the group now sequenced entire genomes (not just fragments) from four samples, all collected in the same area, and on those sequences ran their genetic analyses again. Again they found the new SIV
_cpz
shockingly similar to HIV-1 group M. The similarity was so close as to leave almost no chance that any other variant, yet undiscovered, could be much closer. Hahn’s lab had located the geographical origin of the pandemic: southeastern Cameroon.

12

S
o much for
where
as well as
when.
AIDS began with a spillover from one chimp to one human, in or near that small southeastern wedge of Cameroon, around 1908 (give or take a margin of error). From there it grew, slowly but inexorably, from a spillover to an outbreak to a pandemic. That leaves our third question:
how?

The Keele paper appeared in
Science
, on July 28, 2006, under the title “Chimpanzee Reservoirs of Pandemic and Nonpandemic HIV-1.” In addition to Brandon Keele as first author, there was the usual list of coauthors, including Mario Santiago, Martine Peeters, several partners from Cameroon, and last again, Beatrice H. Hahn. The data were fascinating, the conclusions were judicious, the language was careful and tight. Near the end, though, the authors let supposition fly:

We show here that the SIV
_cpz
Ptt
strain that gave rise to HIV-1 group M belonged to a viral lineage that persists today in
P. t. troglodytes
apes in southeastern Cameroon. That virus was probably transmitted locally. From there it appears to have made its way via the Sangha River (or other tributaries) south to the Congo River and on to Kinshasa where the group M pandemic was probably spawned.

But the phrase “transmitted locally” was opaque. What mechanism, what circumstances? How did those crucial events occur and proceed?

Hahn herself, along with three coauthors, had addressed that back in 2000, when she first argued the idea that AIDS is a zoonosis: “In humans, direct exposure to animal blood and secretions as a result of hunting, butchering, or other activities (such as consumption of uncooked contaminated meat) provides a plausible explanation for the transmission.” She was alluding to the cut-hunter hypothesis. More recently she addressed it again: “The likeliest route of chimpanzee-to-human transmission would have been through exposure to infected blood and body fluids during the butchery of bushmeat.” A man kills a chimpanzee and dresses it out, hacks it up, in the course of which he suffers blood-to-blood contact through a cut on his hand. SIV
_cpz
passes
across the species boundary, from chimp to human, and taking hold in the new host becomes HIV. This event is unknowable in its particulars but it’s plausible, and it fits the established facts. Some variant of the cut-hunter scenario, occurring in a forest of southeastern Cameroon around 1908, would account not just for Keele’s data but also for Michael Worobey’s timeline. But then what? One man in southeastern Cameroon is infected.

“If the spillover occurred there,” I asked Hahn, “how was it that the epidemic began in Kinshasa?”

“Well, there are lots of rivers going down from that region to Kinshasa,” she said. “And the speculation, the hypothesis, is that is how the virus traveled—in people, not in apes. It wasn’t the apes that got into the canoe for a little visit of Kinshasa. It was the people who carried the virus down, most likely.” Sure, she acknowledged, there was a slim chance that someone might have brought a live chimp, captive, infected, all the way down from the Cameroonian wedge—“but I think it is highly unlikely.” More likely the virus traveled in humans.

Sexual contacts in the villages kept the chain of infection alive, though barely, by this line of speculation, and the disease didn’t explode as a notable outbreak—not for a long while. When someone died of immunodeficiency, the death may have seemed unremarkable amid all other sources of mortality. Life was hard, life was perilous, life expectancy was short even apart from the new disease, and many of those earliest HIV-positive people may have succumbed to other causes before their immune systems failed. There was no epidemic. But the chain of infection sustained itself. Each HIV-positive person infected, on average, at least one other person. The virus seems to have traveled just as people traveled in those days: mainly by river. It made its way out of southeastern Cameroon along the headwaters of the Sangha River, then down the Sangha to the Congo, then down the Congo to Brazzaville and Léopoldville,
the two colonial towns on either side of a huge broadening of the river, which was then known as the Stanley Pool. “Once it got into an urban population,” Hahn said, “it had an opportunity to spread.”