Spillover: Animal Infections and the Next Human Pandemic (52 page)

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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S
creening 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 had these strains of virus grown apart? 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, as I’ve explained, 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, SIV
cpz
. 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.

So the spillover had occurred by 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 Jean-Jacques Muyembe, Jean-Marie Kabongo, and Dirk Teuwen, 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 it was only part of Worobey’s contribution. Placing the crucial spillover in time represented a big step toward understanding how the AIDS pandemic may have started and grown.

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P
lacing 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 got 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-1 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-1 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 mentorship 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 chimpanzee 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 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 led 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, of 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 can 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 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 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, 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 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 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.

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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, 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 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 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 SIV
cpz
.