“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 sample didn’t match quite so closely with HIV-1 as Feng Gao’s had done, and it came from an individual of a different subspecies, the eastern chimp (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, nonhabituated 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 the central chimpanzee, the animal 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 central chimpanzees (though a couple dozen derived from chimps belonging to a different subspecies, P. t. vellerosus, which range just north of a major river). Keele then looked for evidence of virus. The samples yielded two surprising results.
95
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, inside the same fence as USAMRIID, where Kelly Warfield had worked on Ebola and, after her accident, spent three weeks in the Slammer. This time, 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 the 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 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, its neighbor to the southeast. The wedge was a hotspot for SIVcpz.
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 SIVcpz 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, ever 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 SIVcpz 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.
96
So much for where as well as when. AIDS began with a spillover from one chimp to one human, in southeastern Cameroon, no later than 1908 (give or take a margin of error), and grew slowly but inexorably from there. 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.” Brandon Keele was first author, with 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 SIVcpzPtt 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. SIVcpz passes across the species boundary, from chimp to human, and taking hold in the new host becomes HIV-1. 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 the Keele data but also for the timeline of Michael Worobey. 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 immune deficiency, 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. R0 remained greater than 1.0. 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, then down the Sangha to the Congo, then down the Congo to Brazzaville and Léopoldville, the two colonial towns on either side of what then was still known as the Stanley Pool. “Once it got into an urban population,” Hahn said, “it had an opportunity to spread.”
But still it moved slowly, like a locomotive just leaving the station. Léopoldville contained fewer than ten thousand people in 1908, and Brazzaville was even smaller. Sexual mores and the fluidity of interactions were unlike what prevailed in the boondocks, but not yet so unlike as they would become. R0 for the virus must have continued to hover around 1.0. Time passed and more people drifted into the towns, drawn by the prospect of working for wages or selling their goods. Habits and opportunities changed. Women came as well as men, though not so many of them, and among those who did, more than a few entered the sex trade.
By 1914, Brazzaville contained about six thousand people and was “a hard mission field,” according to one Swedish missionary, where “hundreds of women from upper Congo are professional prostitutes.” The male population included French civil servants, soldiers, traders, and laborers, and they probably outnumbered females by a sizable margin, due to colonial policies that discouraged married men, coming there to work, from bringing their families. That gender imbalance heightened the demand for commercial sex. But the format for bought favors, in those early years, was generally different from what the word “prostitute” might suggest—grindingly efficient, wham-bam encounters with a long succession of strangers. Instead there were single women, known as ndumbas in Lingala and femmes libres in French, “free women” as distinct from wives or daughters, who would provide their clients with a suite of services, ranging from conversation to sex to washing clothes and cooking. One such ndumba might have just two or three male friends who returned on a regular basis and kept her solvent. Another variant was the ménagère, a “housekeeper” who lived with a white colonial official and did more than keep house. Commercial arrangements, yes, but these didn’t represent the sort of prodigiously interconnected promiscuity that could cause a sexually transmitted virus to spread widely.
Across the pool in Léopoldville, meanwhile, the disparity of genders was even worse. This town was essentially a labor camp, controlled by its Belgian administrators, inhospitable to families, where the male-female ratio in 1910 was ten to one. Travel through the countryside and entry into Léopoldville was restricted, especially for adult females, though some women managed to get false documents or evade the police. If you were a restless, imaginative girl in one of the villages, poorly fed and poorly treated, to be a ndumba in Léopoldville could well have seemed enticing. Here too, though, even with ten horny men for each woman, commercial sex didn’t happen in brothels or by streetwalking. Free women had their special friends, their clients, maybe several contemporaneously, but there was no dizzying permutation of multiple sexual contacts, not yet. One expert has called this “a low-risk type of prostitution,” with regard to the prospects of HIV transmission.
Léopoldville also supported a lively market in smoked fish. Ivory, rubber, and slaves were traded there, for export, with profits going mainly to white concessionaires, well into the colonial era. Although a deep canyon and a set of forbidding cataracts stood between the Stanley Pool and the river’s mouth, isolating both cities from the Atlantic, a portage railway built in 1898 breached that isolation, bringing more goods and commerce, which brought more people, and in 1920 Léopoldville replaced a downriver town as capital of the Belgian Congo. By 1940, its population had edged up to forty-nine thousand. Then the demographic curve steepened. Between 1940 and independence, which came in 1960, the city grew by almost an order of magnitude, to about four hundred thousand people. Léopoldville became Kinshasa, a twentieth-century African metropolis, where life was very different from what passed in a Cameroonian village. The tenfold population increase, along with the concomitant changes in social relations, might go a long way to explain why HIV “suddenly” took off. By 1959, the ZR59 carrier was infected, and a year later in the same city the carrier of DRC60 too. By that time the virus had proliferated to such a degree, mutating and diversifying, that DRC60 and ZR59 represented quite different strains. R0 now must have well exceeded 1.0, and the new disease spread—through the two cities and eventually beyond. “You know,” Hahn said, “a virus was at the right place at the right time.”
When I read Keele’s presentation of the chimp data and the analysis, in early 2007, my jaw dropped like a pound of ham. These folks had located Ground Zero, if not Patient Zero. And when I looked at the map—Figure 1 in Keele’s paper, showing the Cameroonian wedge and its surroundings—I saw places I knew. A village where I had slept. A river I had ascended in a motor pirogue. Turns out that, during my journeys with Mike Fay across the Congo basin, seven years earlier, besides footslogging through Ebola country we had also passed very near the cradle of AIDS. After talking with Beatrice Hahn, I thought it might be illuminating to go back.
97
We rode east from Douala in a beat-up but sturdy Toyota truck, leaving at dawn, getting ahead of the crush, our gear stashed under tarps in the pickup’s bed. Moïse Tchuialeu was my driver, Neville Mbah my Cameroonian fixer, and Max Mviri, from the Republic of the Congo, was along to handle things when we reentered his country in the course of the crazy loop I had planned. Max and I had flown up from Brazzaville the night before. We were a genial foursome, eager to move after the hassles of preparation, rolli
ng past the closed shops and the billboards to the city’s eastern fringe, where traffic thickened in a haze of blue diesel exhaust and the outlier markets were already open for business, selling everything from pineapples to phone minutes. Highway N3 would take us straight to Yaoundé, Cameroon’s capital, and then another big two-lane onward from there.
During a stop in Yaoundé, around midday, I met with a man named Ofir Drori, head of an unusual activist group called LAGA (the Last Great Ape Organization) that helps government agencies in Central Africa to enforce their wildlife-protection laws. I wanted to see Drori because I knew that LAGA was especially engaged on the problem of apes being killed for bushmeat. I found him to be a lean Israeli expat with dark, alert eyes and a patchy goatee. Wearing a black shirt, black jeans, a black ponytail, and an earring, he looked like a rock musician or, at least, a hip New York waiter. But he seemed to be a serious fellow. He had come to Africa as an adventure-seeking eighteen-year-old, Drori told me, and gotten involved with human-rights work in Nigeria, then moved to Cameroon, did a little gorilla journalism (or was it guerrilla journalism?), and became a passionate antipoaching organizer. He founded LAGA, he said, because enforcement of Cameroon’s antipoaching laws had been terrible, nonexistent, for years. The group now provides technical support to investigations, raids, and arrests. Subsistence hunting for duikers and other abundant, unprotected kinds of animal is legal in Cameroon, but apes, elephants, lions, and a few others are protected by law—and increasingly by actual enforcement. Perpetrators are finally getting busted, even doing time, for dealing in ape flesh and other contraband wildlife products. Drori gave me a LAGA newsletter describing the efforts to stem poaching of chimps and gorillas, and he warned me against the myth that ape hunting is a problem because local people are hungry. The reality, he said, is that local people eat duikers or rats or squirrels or monkeys—if they eat meat at all—whereas the fancy stuff, the illicit delicacies, the chimpanzee body parts, the gobs of elephant flesh, the hippopotamus steaks, get siphoned away by upscale demand from the cities, where premium prices justify the risks of poaching and illegal transport. “What brings the money are the protected species,” he said. “Things that are rare.” It sounded like the Era of Wild Flavor back in southern China.
Spillover Page 44