The Origins of AIDS

by Pepin

  Chimpanzees are largely diurnal. To sleep at night, each individual builds a nest in a tree, complete with a pillow, 9–12 metres above the ground, which is normally used only once. For this reason, scientists have used nests to estimate chimpanzee populations, based on counts by surveyors who walk on line transects through forested areas as a sampling method. Population density of P.t. troglodytes is generally between 0.1 and 0.3 km2. Most communities live in forested areas, and a minority in savannahs.

  Chimpanzees are intensely territorial and most troops spend their entire lives within a 20–50 km2 area. Adult males are aggressive, and spend much of their time patrolling their small territory. Males of one troop can form raiding parties to attack lone males (or couples) from other troops. P.t. troglodytes chimpanzees usually have a hostile and violent attitude towards members of other communities. Among their P.t. schweinfurthii counterparts in Tanzania, primatologists documented a war between two neighbouring communities which, after three years of attacks and killings, ended with the complete annihilation of the weaker troop.1,11–13

  P.t. troglodytes chimpanzees are able to develop and use tools, mostly sticks to procure food (for instance, to dig out ants or termites or extract honey from hives). Unlike gorillas, chimpanzees are omnivorous, with a highly diversified diet consisting mostly of fruits, leaves, seeds, plants, insects and eggs, but they occasionally eat vertebrates, including monkeys, antelopes and warthogs.

  An infant chimp spends the first five years of its life completely dependent on its mother. Like humans, they become progressively autonomous during adolescence, reaching sexual maturity at age 12–13. Chimpanzees are promiscuous, and most of their sexual activity takes place when the adult female is in heat and her vulva swells, which attracts the males, who copulate with her quickly, one after the other. As many as six different males may copulate with the same female in just ten minutes. Some males establish an exclusive relationship with a female of their choice, presumably for reproductive purposes, and take her on a ‘honeymoon’ far from the other chimpanzees. This usually only lasts for a week or two during which they copulate as often as five times a day. So while their behaviour limits the transmission of pathogens between troops, sexually transmitted infectious agents will easily disseminate within a given troop once they have been successfully introduced.

  P.t. troglodytes chimps have low fertility: on average, 800 matings occur for each conception. During their reproductive years (from age 14 to 40), females give birth to a mean of 4.4 babies, half of which die before reaching maturity. Each female has a lifetime reproductive success of only 2.3. A small increase in mortality, due to hunting or diseases, is sufficient to reduce this number to less than two and for the population to contract.14,15

  Like humans, chimpanzee communities are occasionally stricken by epidemics. In Gombe, during an outbreak in the region’s human population, poliomyelitis caused four deaths and left some chimpanzees permanently paralysed. Respiratory infections followed, also with fatal consequences. This reflects not just the communal nature of life among the chimpanzees, which have frequent and close contacts with other members of their troop, but also their biological similarity to humans, whose microbes can be transmitted to chimpanzees and vice versa.1

  All kinds of trees

  We will now examine how it gradually became clear that one subspecies of chimpanzees was the source of HIV-1. But first, let us review quickly a science called phylogenetics. Phylogenetics uses nucleotide sequences to reconstruct the evolutionary history of various forms of life, including microbial pathogens. A ‘phylogenetic tree’ superficially resembles a genealogical tree. However, phylogenetic trees describe the relatedness between living organisms (and their classification) rather than ancestry. They measure the genetic distance between organisms, and identify the nearest relatives. Because ancestors are not available to be tested, ancestry is assumed rather than proven. Each division in the tree is called a ‘node’, the common ancestor of the organisms or the isolates identified to its right. After such branching, the organisms and their sequences evolve independently. The ‘root’ (at the extreme left) is the assumed common ancestor of all organisms in the tree. To construct a phylogenetic tree, molecular biologists compare the differences in nucleotide sequences of many isolates of putatively related organisms. This exercise is repeated for various genes; if the findings are the same for two or three genes, scientists are confident that they have produced the right phylogenetic tree.

  An ‘isolate’ corresponds to a given pathogen obtained from one specific patient or animal at a specific point in time. If substantial laboratory work is done on any isolate, it will be given a name corresponding either to the initials of the patient, the name of the city or country where it was obtained or whatever the researcher decides to call it. Like children’s names, these names serve only one purpose, to distinguish isolates from each other.

  For two isolates belonging to the same species, a greater degree of divergence, corresponding to a larger cumulative number of errors in replication, indicates that their common ancestor was further back in time compared to isolates with a lesser degree of divergence. This is like brothers and sisters, born of the same mother and father, being more similar to each other than distant cousins who only share, say, great-grandparents. In practice, phylogenetic trees tell us that certain viruses are closely related and have a relatively recent common ancestor (these are said to ‘cluster’), like brothers or first cousins, while for other viruses the relationship is similar to that of tenth cousins, whose common ancestors lived many generations ago.

  The first report of the isolation of a simian immunodeficiency virus (SIV) from a chimpanzee born in the wild came in 1989. This isolate, given the name SIVcpz-gab1, was obtained from a chimpanzee kept at the primate centre of Franceville, Gabon, where fifty chimps had been tested with assays used for the detection of anti-HIV antibodies in humans. Only two carried such antibodies; from one of them, the virus could be grown in cell culture, and its proteins were analysed. This chimpanzee, captured at six months of age, was four years old when the blood sample was obtained and seemed healthy despite presenting enlarged lymph nodes. Based on the crude methods available at the time, this SIV isolate was described as related although not identical to HIV-1. Phylogenetic analyses suggested that SIVcpz-gab1 was closer to HIV-1 than to HIV-2 and to SIVs from African green monkeys, mandrills and other monkeys.16–17

  It was not possible to isolate the virus from the second seropositive chimp, a two-year-old animal shot by hunters and that died of its wounds shortly after being brought to Franceville for care. A few years later, thanks to technological advances, nucleic acid amplification was used on this chimp’s lymphocytes (which had been kept frozen), in order to sequence parts of the viral genome. This isolate became known as SIVcpz-gab2. It was phylogenetically close to SIVcpz-gab1. In 1992, a third isolate (SIVcpz-ant) was obtained from Noah, a five-year-old chimpanzee captured in the wild and impounded by customs officers in Brussels upon illegal arrival from Zaire. His isolate was somewhat divergent from HIV-1 and from the two previous SIVcpz isolates.18–20

  In 1999, a fourth isolate, SIVcpz-US, was obtained from Marilyn, caught in the wild in an unknown African country and imported into the US as an infant in 1963. Marilyn was used as a breeding female in a primate facility until she died in 1985 at the age of twenty-six, after delivering still-born twins. During a survey of captive chimpanzees, Marilyn was the only one that was seropositive for HIV-1 antibodies. She had not been used in AIDS research, but had received human blood products between 1966 and 1969. During this early period, it is very unlikely that the blood products contained HIV-1, so there was a good chance that Marilyn had acquired her SIVcpz infection in Africa. SIV sequences were amplified from the spleen and lymph node tissues procured at autopsy. Using mitochondrial DNA analyses, researchers identified the subspecies of chimpanzees from which this recent and the previous three isolates had been obtained.21–22

  As cou
ld have been expected from the geographic distribution of Pan troglodytes subspecies, Noah (from Zaire) was a P.t. schweinfurthii while the other three, including Marilyn, were P.t. troglodytes. As illustrated in Figure 1, phylogenetic analyses revealed that the three SIV isolates obtained from P.t. troglodytes were similar to each other, and similar to HIV-1 strains from humans, while Noah’s SIVcpz-ant diverged from these and lay outside this cluster, as did HIV-2 and SIVs obtained from other non-human primates.

  Figure 1 Phylogenetic analysis showing the relationship between SIVcpz-US and SIVcpz-gab1 obtained from P.t. troglodytes chimpanzees (bold) and isolates from humans infected with HIV-1 (group M, group N, group O). The SIVcpz isolates obtained from P.t. troglodytes cluster within the HIV-1 isolates, while SIVcpz-ant obtained from a P.t. schweinfurthii chimpanzee (italics) lies outside. Other SIV isolates obtained from monkeys and human isolates of HIV-2 lie further away.

  Adapted from Gao.21

  Thus, naturally occurring SIVcpz strains fell into two related but highly divergent, chimpanzee subspecies-specific, lineages: one for P.t. troglodytes and another for P.t. schweinfurthii. It was bravely concluded that P.t. troglodytes was the primary source of HIV-1 group M and its natural reservoir, and that there had been host-dependent evolution of SIVcpz in chimpanzees resulting in P.t. troglodytes and P.t. schweinfurthii being infected with different lineages of SIV. Scientists could not rule out the possibility that other chimpanzee subspecies, especially P.t. schweinfurthii, could have transmitted their viruses to humans. This prudence was justified because a single isolate of SIVcpz from P.t. schweinfurthii was available. It was possible that in the future other isolates of SIVcpz, more similar to the human isolates of HIV-1, might be found in P.t. schweinfurthii. Additional isolates of SIVcpz were later obtained from captive P.t. troglodytes in Cameroon, some of which were similar to those human HIV-1 isolates from the same country, reinforcing the view that HIV-1 originated in chimpanzees.21,23

  Since this initial work was conducted mostly with chimpanzees which had been in captivity for some time, it was questionable whether the apes had acquired their SIVcpz naturally in the wild or artificially in their cages where they had been in contact with other primates. In the first case, the puzzle was close to being solved while, in the second, researchers had ventured down the wrong track. Non-invasive technologies were then developed to measure the presence of SIV antibodies and nucleic acids among chimpanzees living in the wild using urine and faecal samples, since obtaining blood samples was neither feasible nor ethically acceptable (some animals may have been hurt or killed in the process). We can but admire the motivation and expertise of these researchers and especially their trackers, roaming through the forest looking for chimpanzee urine or stools, which they had to distinguish from those of other animals. Urine samples proved inferior to faeces and were abandoned.

  Among 100 wild P.t. schweinfurthii from Uganda and Tanzania, only one was infected with SIVcpz-tan1. This isolate was similar to the previous SIVcpz-ant isolate from Noah, the Zairean P.t. schweinfurthii. More isolates were later found among P.t. schweinfurthii chimps in Gombe, where SIVcpz prevalence was estimated to be around 20%. Phylogenetic analyses showed that these isolates clustered with SIVcpz-ant and diverged from the P.t. troglodytes isolates and from HIV-1 (Figure 2), confirming that P.t. schweinfurthii was not the source of HIV-1. Testing of additional P.t. schweinfurthii chimps from the Budongo forest of Uganda, the Mahale park in Tanzania and the Nyungwe reserve in Rwanda (Map 3) failed to identify a single animal infected with SIVcpz. This heterogeneous distribution of SIVcpz, which has recently been mirrored in a study of P.t. schweinfurthii in the DRC, probably reflects the community structures of chimpanzee populations and their behaviour: they have few contacts with chimpanzees belonging to other communities, except during territorial fights or when adolescent females migrate to other troops. But once SIVcpz is successfully introduced into a community, there seems to be substantial transmission between its members, sexually or otherwise.24–28

  Figure 2 Phylogenetic analysis showing the relatively distant relationship between SIVcpz isolates obtained in Tanzania from P.t. schweinfurthii chimpanzees (italics) and SIVcpz-ant obtained from a P.t. schweinfurthii chimp from the DRC (italics), clearly separated from the HIV-1 group M isolates. The latter are close to SIVcpz isolates obtained from P.t. troglodytes (bold). HIV-1 group O lies outside the other HIV-1 isolates (in contrast to HIV-1 group N, which lies inside).

  Adapted from Santiago.26

  SIV is non-existent among captive P.t. verus (the western chimpanzee), about 1,500 of which were tested and found to be uninfected. Surveys of wild P.t. verus and P.t. ellioti also failed to find a single case of SIVcpz infection. Why is SIVcpz absent within these two subspecies? Presumably, because SIVs were introduced into P.t. troglodytes and P.t. schweinfurthii only after these subspecies had diverged from P.t. verus and P.t. ellioti half a million years ago. Such a scenario would imply that there has been little contact between the subspecies ever since, which is possible since the large rivers of Africa constitute watertight barriers.29

  Prevalence of SIVcpz among wild populations of P.t. troglodytes was then measured in an extraordinary study performed in ten forest sites throughout southern Cameroon. To make sure that the faeces originated from P.t. troglodytes and to avoid counting stools from any individual chimp more than once, the researchers amplified a number of host DNA sequences for species, gender and individual identification. In other words, they used the chimpanzee cells present in stools to fingerprint molecularly each and every individual ape who had defecated. After excluding degraded specimens, those that contained gorilla (the trackers’ noses may not always be perfect!) or P.t. ellioti DNA, specimens were available from 106 individual P.t. troglodytes chimpanzees. Sixteen were infected with SIVcpz. Again, there was a lot of variation in SIVcpz prevalence between the study sites: in four of them not a single infection was found; in three sites prevalence was over 20% and the highest was 35%.25

  Phylogenetic analyses (Figure 3) showed that all sixteen new SIVcpz isolates were closely related to SIVcpz isolates from captive P.t. troglodytes chimps and to HIV-1 groups M and N, but not to HIV-1 group O (always the outlier) or SIVcpz obtained from P.t. schweinfurthii. This phylogenetic proximity confirmed – now irrefutably – that the SIVcpz of P.t. troglodytes of central Africa was indeed the source of HIV-1 group M. Game over for this part of the story.

  Figure 3 Phylogenetic analysis showing the relationship between SIVcpz from P.t. troglodytes chimpanzees in Cameroon or Gabon (bold) and isolates from humans infected with HIV-1 group M, HIV-1 group N and HIV-1 group O. SIVcpz isolates obtained from P.t. troglodytes cluster with the HIV-1 group M and N isolates, while HIV-1 group O remains an outlier. SIVcpz obtained from P.t. schweinfurthii chimpanzees in Tanzania or DRC (italics) lie further away.

  Adapted from Keele.25

  Chimpanzee populations separated by long distances or natural barriers like rivers harboured distinct lineages while adjacent troops harboured viruses closely related to each other. More detailed analyses of the genome showed strong clustering of human HIV-1 groups M and N viruses with the SIVcpz lineages obtained from some specific P.t. troglodytes troops in southern Cameroon. In other words, in these rural communities, the local strains of HIV-1 infecting humans genetically resembled the local strains of SIVcpz from the chimpanzees living close by. The SIVcpz isolates from south-east Cameroon, towards the border with Congo-Brazzaville and the Central African Republic, were most closely related to HIV-1 group M, while those from south-central Cameroon were closer to HIV-1 group N.30

  Additional faecal samples from P.t. troglodytes were collected over the following years, mostly in Cameroon, where the prevalence of SIVcpz infection is now estimated to be 5.9%, a figure that I will use for calculations in forthcoming chapters. In the Central African Republic, no SIVcpz infection was found but fewer than fifty specimens have been tested.31

  SIV infection was found among faeces from west
ern gorillas (Gorilla gorilla gorilla); a virus which was called SIVgor. SIVgor is very similar to HIV-1 group O, rather than to group M. Thus gorillas are not the source of the HIV-1 group M pandemic. Without getting into the details, chimpanzees may be the source of HIV-1 group O as well, which they transmitted to humans and to gorillas independently, or to gorillas first, which then infected some humans.31–33

  Until proven otherwise, it is most likely that the modes of transmission of SIVcpz between chimpanzees are the same as in humans: sexual intercourse, from mother to child and possibly through blood–blood contacts. There is much sexual promiscuity in chimpanzees. For instance, one adult male in Gombe is known to have mated since puberty at least 333 times with 25 different females, and of course only a very small proportion of all matings can be observed. A female called Flo was once observed to copulate fifty times within a twenty-four-hour period. The substantial genital swelling of females during oestrus may facilitate transmission of viruses by making the mucosa more fragile. Most of this sexual activity takes place within the closely knit community. A study of paternity among chimpanzee communities showed that only 7% of offspring had a father from outside the troop. Transmission between troops could occur via out-migration of adolescent females, or during fights between males when blood-borne viruses could be exchanged.3,11,12

  The fourth ape

  A weakness in the investigations of SIV among chimpanzees is the dearth of virological information about the fourth ape, the Pan paniscus bonobo. Previously called the pygmy chimpanzee, this was a misnomer since the difference in size compared to Pan troglodytes is minor. It inhabits parts of the DRC south of the Congo but north of the Kasaï–Sankuru river system, in the Congo central basin which has low human populations but is linked by rivers to Léopoldville–Kinshasa, the main market for its farming and fishing products.