SCIENCE: AIDS: THE ROOTS OF A VIRUS

Using new techniques of molecular epidemiology, biologists are trying to trace HIV back to its origins. They may soon be able to draw the virus's evolutionary tree
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IT IS a truth universally acknowledged among scientists that they have been extraordinarily fortunate in the timing of the Aids epidemic. Had HIV made its appearance in the middle rather than at the end of the 20th century, then the genetic technology needed to identify the virus - let alone study it - would still not have been invented. It is a small consolation but one that holds out the promise of answers to the questions: why did HIV emerge now, and where did it suddenly come from?

Over the past few years a new branch of evolutionary research is beginning to draw a picture of the early history of HIV. Called molecular epidemiology, it attempts to match what is known about the course of the epidemic to what can be gleaned from the genetic relationships between the many different strains of HIV found in the world today. The end result is a detailed family tree of the virus that enables scientists to understand more fully how the epidemic spread from one person to another, one group to another and one country to another.

More intriguingly, the tree permits scientists to look back in time to the points where one type of virus branched off from another. It is another universal truth in biology that related life forms have a common ancestor, and the closer the relationship, the more recent the common ancestor. Using this principle, evolutionary biologists are studying the complex family tree of HIV with the aim of shedding light on the earliest common roots of the virus. Eventually, they believe, they may be able to answer the questions of when, where and how HIV originated. They may even be able to solve the biggest conundrum of all - why now?

The story of Aids usually begins in the bath houses of California in the early 1980s when the disease was first diagnosed among promiscuous gay men. Scientists from the Pasteur Institute in Paris first isolated HIV in 1983 and a year later researchers at the US National Cancer Institute confirmed the discovery. Finding the virus meant scientists could compare it against similar viruses infecting other animals - in the hope of shedding light on common ancestors, and the origins of the human immunodeficiency virus.

An initial genetic analysis soon confirmed that HIV belonged to a particular class of microbe called the lentiviruses. At that time its closest known relatives were lentiviruses that infected horses and sheep, which although similar in many respects to HIV were thought to be too distant genetically to be contenders for the immediate ancestor of the human virus. It is perhaps no accident that failing to find the ancestral animal virus for HIV coincided with some wild speculations about the origins of Aids, notably that it was a KGB or CIA plot.

Finally, in the mid 1980s, scientists found a much closer relative of HIV in monkeys. They called it simian immunodeficiency virus, SIV, and the immediate thought was that an early form of SIV had somehow ''jumped the species barrier'' from monkey to man. Quite how or why this event occurred nobody was sure but it was, and still is, a reasonable assumption given that other human viruses - such as influenza - also spend much of their time living in animal hosts. There is now abundant evidence that SIV is much older than HIV, which neatly fits this theory. SIV has probably infected some species of African monkeys for thousands of years.

What was initially confusing about SIV was that it was found in both captive macaque monkeys (which normally live in Asia) and captive African green monkeys, which live on a quite separate continent. Eventually it became apparent that wild macaques and other Asian monkeys did not have SIV. Captive macaques had clearly become infected by being confined with African monkeys, which seemed to be the natural ''reservoirs'' of SIV in the wild. This was one of the strongest arguments for an "out of Africa'' origin of the human virus. This theory gained credibility when it emerged that HIV was prevalent in central Africa.

An important development in the Aids story took place when French and Portuguese scientists isolated a highly unusual strain of HIV from patients who had lived in west Africa. For some reason the usual antibody tests for HIV did not work on these patients. They appeared to be ''HIV negative'' yet still had the symptoms of Aids. When the scientists managed to isolate the HIV from the infected tissues of these people they found its genetic structure was rather different to HIV found elsewhere in the world, which is why the antibody test failed to work. They felt the discovery warranted giving this virus a distinct name, and so called it HIV-2. The ''original'' HIV became known as HIV-1.

To this day all Aids viruses fall into one of these two groups. Type 1 is found throughout the world and accounts for the overwhelming majority of Aids cases. The much rarer Type 2 is centred mostly in west Africa - or in people who have had close connections there.

What has really excited evolutionary biologists about HIV is the same trait that makes it such a difficult opponent. It evolves at an incredible speed. The genetic structure of the virus - the sequence of chemicals that determine the make-up of its genes - changes about a million times faster than that of humans. Along with the influenza virus, HIV is the fastest evolving life form on the planet. This makes it difficult for anti-viral drugs to work against it, because HIVs within the body eventually mutate into forms that are drug-resistant. For the same reasons, it is practically impossible to design an effective vaccine against such a moving target.

However, a virus that evolves so quickly generates hundreds, if not thousands of slightly different strains during the course of infecting a single individual. A person can be infected with one strain of HIV and years later die with an entirely different population of HIV ''quasispecies'' - a collection of strains each with slightly different genetic sequences to the rest. On a wider scale, this rapid evolution means that in the world at large there is a huge variation in HIV-1s. It is this almost unimaginable variation that is providing molecular epidemiologists with the raw data to draw a family tree.

A tree of relationships, however, does not on its own allow them to look back in time to the origins of the virus. For this they need to know how fast the ''molecular clock'' of evolution is ticking. Each tick of the molecular clock results in a change in the genetic sequence. Paul Sharp, professor of genetics at the University of Nottingham, was one of the first to assess the speed at which the molecular clock of HIV is running. His estimate that it is ticking about a million times faster than the molecular clock dictating the speed of human evolution has since been supported by other scientists.

The clock is crucial to looking back to the early years of HIV's evolution, he says. ''There does not have to be a molecular clock to draw up a tree, but there would have to be a molecular clock for you to put times on the branching points of the tree.'' In simple terms, knowing the variation of a group of present-day HIVs and knowing the ''ticking'' rate of the clock, you can calculate how long it has taken for all that variation to occur. The end result is that you know pretty accurately when the common ancestor of this group existed.

Applying the technique gives some interesting results. Take for instance the main group of HIV-1s, called the ''M'' group. This group can be split into a number of subtypes, ranging from subtype A to subtype I. The molecular clock posits that the common ancestor of all the subtypes of the M group existed about 1960. This is remarkable given that the vast majority of all HIV in the world today falls within the M group of HIV-1. This means the huge explosion in variation in the Aids virus has occurred within a period of about 30 years, a mere blink of the eye on the evolutionary timescale.

This brings scientists closer to answering the difficult question of why did Aids and HIV emerge now, in the second half of the 20th century? One view is that it had emerged several times before in history as a result of an SIV jumping from monkey to man, but because human populations in Africa were so isolated, the virus did not get beyond a certain close- knit group. With the huge demographic upheavals in Africa from the 1950s on, and the growth of world travel, all this changed. (An intriguing, if unsubstantiated, alternative view is that an SIV jumped from monkey to man because at the end of the 1950s there was a huge growth in the trade in African monkeys for scientific research, which would have increased the chances of virus transmission.)

Since HIV emerged 30 years ago, what has been staggering has been the speed at which it has spread and evolved. Undoubtedly the rapid increase in variation of HIV-1 over that period has much to do with the equally rapid spread of the virus, first in Africa and then throughout the world. As the virus infected a greater pool of people, it exploited its new ''habitat'' by generating the great diversity that gave rise to all the subtypes present today. But this still does not explain why this happened.

Most investigators who have studied the evolution of the ''M'' group of HIV with all its subtypes around the world today are agreed that the dramatic social and political upheavals in Africa and elsewhere led to the rapid spread of the virus around the globe. Mass movements of people from the rural heartlands of Africa to the fast-growing cities and trans- continental roads that carried goods, people, the sex industry and HIV across Africa also played their part. On top of this, a huge explosion in global tourism meant that there were few places on Earth that were sufficiently isolated from the emergence of a new infectious agent.

This seems the most plausible explanation for how HIV spread as rapidly as it did, but it is more difficult to answer the question of why it emerged at all. However, the strongest clue yet comes from looking at the genetic relationships between HIV-2 and a type of SIV found in sooty mangabey monkeys, which live wild in the area of west Africa where HIV-2 is found. The genetic sequence of the sooty mangabey SIV is practically indistinguishable from some HIV-2s.

Evolutionary biologists believe that there is now convincing evidence that this monkey virus - present in an animal that is routinely captured and butchered for meat by West Africans - ''jumped'' from monkey to man on at least five separate occasions, perhaps during the mingling of human and monkey blood when the animals were slaughtered. ''It looks to me as if the different subtypes of HIV-2 represent different instances of the mangabey virus crossing into humans,'' Professor Sharp says. (However, there is as yet not enough research on the molecular clock of HIV-2 to predict when these events occurred.)

Could the same scenario have happened for HIV-1? Professor Sharp and colleagues believe it could, but they face one important difficulty. The simian virus found in African green monkeys is clearly the strongest contender for an ancestral natural reservoir of HIV-1, given that the green monkey virus is so prevalent in the wild, just like the SIV of sooty mangabeys. The problem, however, is that its genetic structure is just too different from HIV-1 to be the immediate ancestor of the human virus.

The only other SIV that comes near HIV-1 is a virus recently found in chimpanzees, the closest living relative of humans. But just four have been identified with SIV and extensive testing of wild chimps has failed to find the sort of prevalence of the monkey virus that is needed for it to be the ancestral reservoir for HIV-1.

This leaves the evolutionary biologists with a conundrum. They are firmly of the opinion that a simian virus must have been the ancestor of HIV, especially as the SIV of African green monkeys is so much older than the human virus. The problem is that there does not yet seem to be a simian immunodeficiency virus - and a suitable host monkey - that can fill the same role as the sooty mangabey virus appears to have played in the origin of HIV-2.

Perhaps there is an as yet undiscovered SIV in a species of small monkey that can take on this mantle. Perhaps, Professor Sharp argues, this species of small monkey lives alongside and is hunted by chimps (as well as humans), which is why there are a few individual chimps infected with the virus. Unfortunately, no scientist has yet found this virus and its monkey host.

One final analysis of the Aids virus family tree reveals another, less numerous group of HIV-1s. Called the ''O'' group, it was only discovered relatively recently in people who live in Cameroon. Professor Sharp and his colleagues believe the O group is genetically so distinct from the M group that it probably came about as a separate monkey-to-human transmission of a simian virus. This double jump from monkey to man that gave rise independently to the two groups of HIV-1s could have occurred as recently as the 1950s. The jury is still out on what was responsible for these two events.

The new science of molecular epidemiology has yet to solve the biggest mystery of all - why now? - though it's getting closer all the time. !

THE ROUTES OF THE AIDS VIRUS

The new science of molecular epidemiology would not be possible without the sophisticated technology for easily deciphering the genetic information of HIV. At the heart of the research is the polymerase chain- reaction test, which can repeatedly amplify minute quantities of the genetic blueprint - DNA - to make many millions of copies until there is enough material to analyse with the technology of gene sequencing.

Such sequencing, which reduces each virus to its basic genetic code, can reveal the unique genetic signature of each strain of HIV. Each particular HIV can be likened to an enormously long ''word'' composed of more than 6,000 ''letters''. However, instead of there being 26 different letters in the genetic alphabet, it consists of just four. Even so, the potential number of viral variants or ''words'' that this can create is enormous. HIV appears to exploit this huge potential for genetic variation with devastating effect.

It is this ability to alter its genetic make-up - and with it to change the nature of the protein coat that prevents the body's immune defences from attacking it - which makes the Aids virus such a formidable target for vaccines and drugs.

By far the most numerous kind of HIV in the world is the main or ''M'' group within the HIV-1 type (see main article). This group is also the most diverse, and scientists have so far been able to categorise at least nine different subtypes within it, labelled subtype A, B, C and so on. Genetic sequences of the members of each subtype are different enough to warrant their belonging to different strains but similar enough to be categorised under the same subtype name.

When a geographical analysis is done of different subtypes, some interesting facts emerge. Subtype B, for instance, predominates in the US and Europe. Scientists believe this shows that this particular subtype was the one that gained a foothold in the US during the late 1970s and then spread quickly to cause the epidemic there and in Europe.

Two different subtypes have spread in Thailand. One appears to predominate in drug users and another in prostitutes, which suggests there are two parallel epidemics taking place. A similar, dual subtype epidemic is happening in Brazil, where scientists have found subtypes B and F in abundance. Paul Sharp and colleagues have also found people infected with both subtypes and now have evidence that this can result in the formation of hybrid HIVs - bringing a new level of genetic diversity into the Aids equation.

The geography of where a particular subtype is found shows how the virus has passed from one country to another. Subtype G, for instance, is found only in the Central African Republic and in one small town in southern Russia, where it is responsible for a small-scale epidemic among about 300 people. This, it has transpired, resulted from a single Russian diplomat who picked up the virus while stationed in central Africa. Another curious finding is that subtype F, found in Brazil and central Africa, has also emerged as a dominant form of HIV in Romania.

A feature of analysing the distribution of HIV subtypes in the world is that it once again underlines the African roots of the virus. Although different subtypes are prevalent in different parts of the world, all can be found in the sub-Saharan countries of central Africa. Whatever the subsequent history of HIV, it must have begun here. SC

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