WHAT CAUSES AIDS? It's An Open Question (June 1994)

THE ISOLATION QUESTION

By Paul Philpott

Reappraising AIDS, June, July, Aug. 1997

Does HIV exist? Do HIV tests indicate HIV infections? Here's why some scientists say no. How an Australian biophysicist and her simple observations have taken center stage among AIDS reappraisers.

Of course HIV exists--I've seen pictures of it in text books and on the news--and scientists work with it every day. How could there be HIV tests if there's no HIV? What those tests detect, that's HIV...

So goes the typical response from physicians, biologists, and AIDS activists when faced with a very simple question: Does HIV exist? But like all questions fundamental to the HIV/AIDS model, nobody asked this in 1984, the year Robert Gallo published a group of four papers in Science (224:497-508, May 4) proclaiming the existence of a unique retrovirus, HIV, that causes AIDS.

Gallo's HIV-AIDS model stood unquestioned in the medical literature for three years, until 1987, when UC-Berkeley retrovirologist Peter Duesberg published the first academic paper contesting the notion of pathogenic retroviruses (Cancer Research 47: 1199-1220). Although disputing the infectious AIDS model, Duesberg accepted Gallo's claim of having prepared isolates of a unique retrovirus, HIV, and having abstracted from them proteins needed to construct tests for identifying people and cells infected with it.

By 1987 the plasma and T4 cells of thousands of AIDS patients had been tested for evidence of the proteins and genetic material from Gallo's "isolates." The AIDS reappraisal movement grew out of Duesberg's critique of these data. HIV exists, but the blood contains so little of it, and it infects so few T4 cells, and replicates--harmlessly--in vitro with so much difficulty, and so many patients test negative for it altogether, that it is just too ineffectual, inactive, and imperfectly correlated with AIDS to explain AIDS.

Out of Australia: Questioning HIV's existence

Before Duesberg's 1987 paper made it to press, a second academic, authoritative deconstruction of HIV had already been submitted for publication in another journal. This one was written by Eleni Papadopulos-Eleopulos, a medical physicist at Australia's Royal Perth Hospital. In 1988 France's Medical Hypotheses (25:151-162) published her paper, "Reappraisal of AIDS: Is the Oxidation Induced by the Risk Factors the Primary Cause?" Papadopulos had independently reached many of Duesberg's conclusions, but ultimately had quite a different take on Gallo's claims: "Unlike other viruses [HIV] has never been isolated as an independent stable particle."

What she meant was this: Electron microscope pictures, micrographs , of samples Gallo calls "HIV isolates"--and of all "HIV isolates" produced before by Luc Montagnier of France, or since by other scientists--show some objects that look like retroviruses (the "HIV") plus lots of other things, including things that clearly aren't viruses. So there's no way to identify the origin of the "HIV" proteins and genetic material abstracted from these samples. Do the proteins come from the objects that look like retroviruses? Or do they represent some of the contaminants?

And what about those retroviral-looking objects? Papadopulos pointed out that among the microbial objects that look like retroviruses are (1) microvesicles: non-infectious, unstable organelles that bud from cells; and (2) endogenous retroviruses: non-infectious, unstable retroviruses coded for by healthy human DNA. She noted that this presents a special problem for the objects called "HIV." They can be observed only in cell cultures that have been stimulated by agents that induce the production of microvesicles and endogenous retroviruses.

Without true isolates of the objects declared "HIV," there really is no way to determine if they constitute what HIV is claimed to be: a retrovirus of exogenous origin (an autonomous entity unaccounted for by a person's inherent DNA library). There is no way to pull proteins and genetic material out of a heterogeneous sample and know that they came from one group of particular looking objects rather than another, or simply from the surrounding molecular soup.

Oxidative stress: Unifying AIDS, its causes, and "HIV"

In addition to introducing an HIV critique based on the principal of viral isolation, Papadopulos also unveiled in her 1988 paper an explanation for AIDS based on the process of oxidative stress. According to Papadopulos, the stimulants used to induce "HIV" phenomena (retrovirus-looking objects plus certain proteins that may or may not be affiliated with those objects) in cultures are oxidizing agents . As are the factors uniting American AIDS patients, including street drugs, hemophilia treatments, and rectally deposited semen. Papadopulos proposed that both "HIV" phenomena and AIDS conditions are consequences of these and other stressors she would introduce in later papers (such as blood transfusions, anti-AIDS pharmaceuticals including AZT, and antibiotics).

Duesberg drew on the 1988 Papadopulos paper (and even earlier writings by John Lauritsen in the gay press) in formulating his 1992 treatise "AIDS Acquired by Drugs and Other Non-contagious Risk Factors" (Pharmacology & Therapeutics 55:201-277). In that paper, Duesberg added to his HIV critique alternative explanations for AIDS. He agreed with Papadopulos that street drugs and hemophilia treatments caused AIDS, but dismissed rectal insemination as inconsequential. His 1992 paper was the first to implicate "anti-HIV" drugs such as AZT, and Papadopulos subsequently adopted them into her oxidative stress model.

That same year, 1992, Papadopulos formed a writing team with two University of Western Australia physician-professors, Valendar Turner of the Department of Emergency Medicine, and John Papadimitriou, Professor of Pathology. Together they published "Oxidative Stress, HIV, and AIDS" (Res-Immunol. 143:145-148), which restated her Unified AIDS Theory.

Virus tests without virus isolation?

In 1993 Papadopulos finally caught the attention of AIDS reappraisers. "Is A Positive Western Blot Proof of HIV Infection?" appeared in Bio/Technology (11:696-707), a major medical journal and sister publication of Nature.

The article debunked the validity of "HIV tests" on several grounds: (1) that they are constructed from the constituents of heterogeneous samples rather than true viral isolates; (2) that proponents of the purported virus (HIV) claim to observe it only in stimulated cultures, as opposed to fresh patient plasma; (3) that accuracies for these tests are established without an independent gold standard (isolation from fresh patient plasma); and (4) that these tests are assumed to be equally accurate for people with and without the risks associated with, and the conditions classified as, "AIDS," a syndrome the purported virus supposedly causes.

Isolation, Papadopulos explains, is the only sure proof that a virus is present--the only direct, unambiguous evidence of a virus. And isolation from uncultured patient plasma is the only sure proof that a person harbors an active infection-- the only sort of infection that can cause disease. She points out that the accuracy for even a properly constructed viral test (one made from true viral isolates) can be established only by answering the following question: In what fraction of people who test positive can the virus be isolated from their fresh (uncultured) plasma?

Instead, "HIV" test accuracies are established using circular logic; "accuracy" for HIV ELISAs is taken as the fraction of positive people who subsequently test HIV Western blot positive. And "accuracy" for HIV Western blot tests is nothing more than reproducibility (the fraction of positive people who test positive when retested).

These pseudo accuracies--each over 99%--are assumed for all people, even those free of the risks and symptoms associated with the syndrome that the purported virus supposedly causes. Yet among risk group members with blood that reacts with these tests--those who test positive--pseudo isolations ("HIV" phenomena in stimulated cultures) are achieved for only some of those with AIDS conditions, and for only a few who are symptom-free.

For example, of risk group members (gay men, drug injectors, and blood recipients) testing "HIV-positive":

(1) Gallo achieved pseudo "HIV" isolations in 26 of approximately 63 (41%) patients with AIDS conditions (this is a generous figure that assumes Gallo's isolations involved only the 88% of his 72 AIDS-diagnosed patients who tested positive) ;

(2) Piatak reported (a) "infectious HIV" (according to some of the same criteria as pseudo isolations) in only 29 of 38 (76%) patients with AIDS conditions and in only two of 21 (10%) patients with no AIDS conditions (Science 259: 1749-1754, 1993); and (b) in one of six (16%) symptom-free patients (Lancet 341: 1099, 1993);

(3) Daar reported "infectious HIV" in none of four symptom-free patients (NEJM 324[14]:961-964, 1991);

(4) Clark reported "infectious HIV" in none of three symptom-free patients (NEJM 324[14]:954-960, 1991); and

(5) Cooper found "infectious HIV" in neither of two symptom-free patients (Lancet 340:1257-1258, 1992).

So among people with AIDS risks, using pseudo isolations from stimulated cultures as an independent standard, HIV antibody tests are between 41% and 76% accurate for people with AIDS conditions, and between 0% and 16% accurate for those with no symptoms, a far cry from the 99% accuracies established using reproducibility and cross-checking.

What about people without AIDS risks? No one has compiled even pseudo isolation data for drug-free, blood product injection-free heterosexuals who test positive. HIV researchers simply assume that the data from risk group studies apply for everyone.

And what about the real accuracy of HIV tests? That is, accuracy established using the only valid gold standard: isolation from fresh plasma. The Australians reason that since isolation from fresh plasma has not been achieved under any circumstance, then the true accuracy for all "HIV tests" should be considered zero , and all positive results should be regarded as false. There is no basis for thinking that a virus observed only in stimulated cultures exists in the plasma of any humans, even those who test positive for it as determined by antibody, antigen, "viral load" or any other assay.

"HIV": Normal cellular residents?

In the Bio/Technology paper, Papadopulos examined what are accepted as substitutes for true HIV isolation. These include "HIV proteins" (gp160, gp120, gp41, p32, p24, and p17), reverse transcriptase, "HIV" DNA and RNA, and retrovirus-looking objects. She suggests that they are each cellular constituents, some normal, some produced in response oxidative stress.

(1) HIV existentialists--those who think HIV exists--hypothesize that gp160 is made of gp120 stuck to gp41, and it decorates HIV, with gp41 embedded in the outer membrane envelope, anchoring gp120, which protrudes outward, ready to latch onto T4 molecules; Papadopulos cites references showing that gp160 and gp120 are oligomers of gp41 (four gp41s stuck together make gp160; three make gp120), and that gp41 might be the ordinary cellular protein actin. (She also cites references showing that cell-free objects considered to be HIV contain no gp120, and thus have no infectious capability, just like endogenous retroviruses.)

(2) The existentialists hypothesize that p17 lines the inside of the envelope, and p24 forms the hollow core; Papadopulos cites references showing that p24 and p17 might be the two constituent globs that form the ordinary cellular protein myosin.

(3) The existentialists hypothesize that p32 decorates HIV's envelope, along with gp160; Papadopulos cites references showing that p32 is the "Class II histocompatibility DR" marker found on all human T immune cells.

(4) The existentialists hypothesize that reverse transcriptase is a constituent of HIV, and is used to make HIV DNA from HIV RNA; Papadopulos cites references showing that this enzyme is a normal constituent of all human cells, and even some ordinary viruses, like hepatitis viruses, which are common in AIDS patients.

(5) Papadopulos shows that no complete "HIV" RNA molecule or DNA genome has ever been identified, that what is claimed to be the "HIV" genome represents bits and pieces of genetic sequences cobbled together, that the "HIV" RNA and DNA haven't been shown to code for what are claimed to be the HIV proteins, and that all the "HIV" genes are very similar to genetic sequences common to all humans.

(6) The existentialists hypothesize that the retrovirus-looking objects in electron micrographs of heterogeneous samples from AIDS patients are identical retroviruses, HIV, that consist of the "HIV" proteins and RNA abstracted from those samples; Papadopulos explains that since those samples are heterogeneous, there's no way to match the retrovirus-looking objects to any material abstracted from the samples, that retrovirus-looking objects are common products of stimulated T-cells, and that such objects are not necessarily viruses of any sort and can be proven to be so only when examined as isolates.

HIV antibodies as autoantibodies

Although the "HIV proteins" haven't been shown to be constituents of a virus, they are the constituents of the ELISA and Western blot antibody tests for HIV. If Papadopulos is correct that these are ordinary cellular proteins, why would humans express antibodies against their own cellular proteins, a condition called autoimmunity ? And why would such antibodies correlate (however imperfectly) with AIDS conditions and AIDS risks?

The Bio/Technology paper argues that antibodies against actin, myosin, and p32 indicate exposure to those proteins donated by other people via injected blood products, unsterile needles, and rectally deposited semen. These factors nearly unify all American AIDS patients, and they are oxidative stressors. So Papadopulos proposes that oxidative stressors cause AIDS conditions and positive HIV tests, thus explaining the correlation between AIDS conditions and positive HIV test results.

(Which is not to say that every positive "HIV antibody" test indicates autoimmunity or oxidative stress, or that autoimmune phenomena always cause disease, or that oxidative stress always causes "HIV" phenomena or AIDS conditions.)

In non-industrial regions such as those in Africa where lots of AIDS patients reside, Papadopulos shows that HIV antibody tests (the only sort of HIV tests used there) cross-react with antibodies against numerous ordinary microbes and parasites that are rampant there due to extremely impoverished living standards. AIDS conditions in these regions, she says, result from those cross-reacting infections, other infections common among impoverished people, and poverty itself.

Proving causation: another need for isolation

Papadopulos' group published another 1993 paper, "Has Gallo Proven The Role of HIV in AIDS?", in the Australian journal Emergency Medicine (5:113-123). This paper presented much of the same data and arguments about the lack of HIV isolation offered in the Bio/Technology paper. But where that paper examined the absolute requirement of viral isolation for constructing and validating viral tests, this paper examined the absolute requirement of viral isolation for demonstrating a causal relationship between a virus and a disease.

The Australians focused here on Gallo's 1984 papers, which they characterized as the most thorough to date. They argued that a virus can only be considered causal for a disease if:

(1) It can be isolated in every case of the disease from fresh (uncultured) plasma. Yet Gallo claimed to isolate HIV only from cultures, and only after stimulation with agents that cause inactive viral DNA (provirus) to produce viruses that might not be present in vivo . Furthermore, Gallo could only claim HIV isolation in 34% of the AIDS patients tested, and even then these claims were based not on real isolation, but on the observation of certain proteins, reverse transcriptase, and retrovirus-looking particles, though usually not all at the same time.

(2) Adding isolates of the virus to cultures of cells of the type affected in the disease in question results in behavior consistent with the disease. In the case of AIDS, that would mean adding HIV isolates to cultures of T4 cells and looking for either cell death (predicted by the original killer HIV model) or high rates of HIV activity (predicted by the new hyperactive HIV "viral load" model). But Gallo found neither. Cells declared "HIV-infected" lived happily ever after, and would produce HIV indicators only when prodded by artificial stimulants.

The Australians emphasized that no researcher since 1984 has improved on Gallo's very weak case for HIV as a cause of AIDS.

All antibodies non-specific

The Bio/Technology paper presented a long list of non-HIV agents that can cause positive reactions on HIV ELISA and Western blot antibody tests. This is very bad news for those tests.

HIV antibody and antigen tests are constructed from heterogeneous samples rather than isolates, and validated against each other rather than the isolation gold standard. Therefore their validity requires that HIV proteins and the antibodies against them be specific . That is, the proteins must be exclusive to HIV, and the antibodies that react with them must react with no other proteins.

Gallo and the other existentialists, Papadopulos explains, simply assume that their "HIV proteins"--and antibodies against them--always indicate a virus made from those proteins, and nothing else. They base this assumption on no data, and no wonder. Only isolation--which none of them has achieved--can demonstrate this sort of specificity. Furthermore, Papadopulos' list of cellular sources for each "HIV protein," and her list of non-HIV entities that cause reactions with "HIV" antibody tests, absolutely falsify the specific antibody ideal for HIV.

False positives

Papadopulos explains that there is no such thing as specific antibodies against any microbial agent. All viral tests (including properly constructed ELISAs and Western blot tests for properly characterized viruses) "cross react" with entities other than their intended targets.

This is why test accuracies must be established for different groups (those with and without symptoms and risks associated with the virus) using the gold standard (virus isolation from fresh plasma).

Properly validated virus tests are not undermined by a list of cross-reacting entities. If the virus can be isolated from the fresh plasma of 99% of the people with certain symptoms who test positive in validation studies, then physicians would have a 99% certainty that a patient with those symptoms who tests positive has an active infection.

The existence of cross-reacting entities becomes important only in circumstances of low accuracy. In the world of properly constructed and validated viral antibody tests, that means symptom-free people, and people who have been exposed to cross-reacting factors.

Virus isolations are rarely achieved in symptom-free people who test positive, which means the accuracy is low for apparently healthy people. The only sensible interpretation for positive results in healthy people is that these people have experienced, sometime in the past, an infection that is no longer active (and is thus inconsequential), or they were exposed to cross-reacting proteins.

Before the introduction of HIV science, physicians did not test healthy people for viral infections, except for people with certain risks, such as recent exposure to someone with a confirmed infection. Validation studies can show a relatively high accuracy for positive tests in symptom-free people with such a risk. So it is rational to test such people. HIV tests are the only viral tests administered routinely to healthy people with no risks.

In the strange case of HIV and AIDS, though, even testing people in the AIDS risk groups is a dubious enterprise. This is because the official risks that define these groups (rectal intercourse, unsterile needle use, blood product injections, residency in impoverished nations), involve exposure to non-HIV factors that cause cross-reactions with these tests.

Virologist Lanka supports Papadopulos

The Bio/Technology paper influenced most reappraisers to question the validity of "HIV" tests, mostly on the grounds of cross-reactivity. Few seemed to appreciate that the isolation question was the real crux of the matter. The question of HIV's actual existence seemed just too big for most reappraisers to tackle. Then along came a young German virologist, Stefan Lanka, co-author of an academic paper that properly established the existence of a marine virus, ectocarpus siliculosis .

The British AIDS reappraisal magazine Continuum published in its April/May 1995 issue Lanka's exposition, "HIV: Reality or Artifact?" This was the first article for a popular audience explaining Papadopulos' contention that HIV simply does not exist, and that the phenomena considered to indicate its presence have non-viral explanations, such as artifacts of the lab procedures applied to cultures made from the blood of AIDS patients. The next issue (June/July) included a fiery and detailed exchange between Lanka and Steven Harris, a physician who advocates the HIV-AIDS model. That article displayed two electron micrographs of properly isolated viruses: Lanka's ectocarpus siliculosis, and adenovirus type 2 (which cause common colds). Those two micrographs exclusively contained identical virus-looking objects. Harris presented a micrograph of what he called an "HIV isolate." Lanka pointed out that this micrograph contained, in addition to retrovirus-looking objects labeled "HIV," lots of microvesicles and "macromolecular debris." Therefore it was not an isolate.

This exchange created such interest--and Continuum 's editor-ial board was so persuaded by Lanka's argument--that the magazine in its January/February 1996 issue posted a 1,000 "Missing Virus Reward" for anyone who could produce a micrograph of a proper "HIV" isolate.

Papadopulos answers the first challenge

In April, 1996, the National AIDS Manual (NAM) Treatment Update published an editorial answering the Continuum challenge. NAM made no claim on the prize, conceding an absence of the micrograph specified by the reward. Instead, NAM argued against the need for such a requirement in establishing the existence of a virus.

Specifically, NAM rejected the Papadopulos/Lanka objections to contaminating material in the available "HIV" micrographs. "...It's like saying that it is impossible to identify a German shepherd dog by its unique appearance," the article reasoned, "if it happens to be surrounded by poodles."

In the May/June issue of Continuum , Papadopulos' team responded to the NAM critique with a remedial lesson in microbiology: "The analogy with HIV is more like someone who does not know what a German shepherd is but who looks at an aerial photograph of a zoo," and notes that some of the objects look like dogs, then "mince[s] up all the objects in the zoo," and presumes to know which teeth, claws, hair, hearts, and stomachs came from the objects that looked like dogs, and claims that those objects are some new breed deserving of a new name.

Instead, German shepherds have been carefully studied on their own, which is why they can be identified merely by their image, even in the midst of other dogs. Certainly a new breed of dog could not be declared--and identified by aerial photographs (the human scale equivalent of an electron micrograph)--without first studying one up-close (the human scale equivalent of viral isolation).

If isolates were obtained of the objects labeled "HIV" in micrographs of heterogeneous samples, and those isolates were shown to consist of a unique, exogenous retrovirus, then there would be a basis for pointing out these objects in heterogeneous samples and declaring them to be "HIV."

Until then, nobody knows what the objects purported to be "HIV" are in any of the "HIV micrographs."

Duesberg demurs, Lanka descries

By the July/August issue, Continuum 's reward had increased to 25,000, and none other than Peter Duesberg wrote in to claim the prize. Conceding that there existed no such micrograph as that sought by Papadopulos and Lanka, Duesberg argued that existing data "exceeded the [Papadopulos/Lanka] criteria" for virus isolation: the isolation of "infectious full length HIV DNA" from "HIV-infected cells," and the detection of this DNA in some T4-cells of nearly 100% of people who test positive for "HIV antibodies," but in nearly 0% of those who test negative.

In the same issue Continuum published rebuttals by both Lanka and the Australian team, which now included a fourth member, David Causer, Senior Physicist at the Department of Medical Physics at the Royal Perth Hospital.

Lanka surprised everyone with his "Collective Fallacy: Rethinking HIV." Leaving it to "the distinguished Australians" to provide "a detailed reply to the Duesberg claim," he leaped past that dialogue and into a novel assertion: all retroviruses are fictions, artifacts of the contrived laboratory conditions invariably used to find them. He described Duesberg as:

limiting his objections to the relatively minor aspect of whether HIV could cause AIDS or not, whereas he really ought to have smelt a rat regarding the whole concept of retroviruses. ...Indeed, the extraordinarily artificial and circumscribed conditions under which reverse transcription could be induced in the laboratory should have alerted everyone to the extreme improbability of such exclusively laboratory conditions having any bearing whatsoever on naturally occurring phenomena .

The Papadopulos treatise

Papadopulos' rebuttal was an exhaustive exposition entitled "The Isolation of HIV: Has It Really Been Achieved? The Case Against," included as a 24-page supplement. She asserted that until a virus has been isolated according to the criteria required by the Continuum reward, its constituents--including genetic material and proteins--cannot be cataloged. So there is no basis for a viral explanation for this correlation.

Yet Duesberg has a point. How can Papadopulos and Lanka explain the high correlation between particular proteins (and antibody reactions to them) and the detection of particular DNA/RNA sequences? This can not be a chance occurrence.

Papadopulos agrees. But she points out that isolating DNA does not equal isolating a virus, and certainly does not "exceed the criteria" specified by the reward, which represent, in fact, an official standard procedure for retroviral identification which was discarded only to accommodate "HIV." Logically, there is no basis for concluding that an RNA molecule abstracted from a heterogeneous sample (even one containing retrovirus-looking objects), or a strip of corresponding chromosomal DNA, originates from a retrovirus. Such an assumption can only apply to RNA abstracted from a retroviral isolate (and only if that RNA is shown to code for the proteins abstracted from the same isolate).

To explain the "HIV" protein-RNA/DNA correlation, Papadopulos referenced studies showing that the correlation between the proteins and the genetic material was not quite as high as in the study Duesberg cited. Then she proposed that the "HIV DNA" in cellular chromosomes might result from the rearrangement (transposition) of a few normal cellular DNA sequences in response to oxidative stress caused by both the AIDS risks (street drugs, etc.) and the laboratory agents required to observe "HIV" phenomena.

Duesberg says this would require an improbable number of nucleic acid rearrangements ("recombinations"), one for each of the 9,150 bases said to constitute the HIV genome. Papadopulos says the number of required rearrangements is actually much lower, since each of the supposed HIV genes are already very similar to recognized normal human genetic sequences.

Is Papadopulos certain that oxidation-induced recombination explains the HIV protein-RNA/DNAcorrelation? No. She's simply convinced that this is more likely than the Duesberg-Gallo explanation, which is that the "HIV" genetic sequences originate in a retrovirus that carries with it the "HIV proteins."

To her, the viral explanation is fatally undermined by several facts: (1) heroic attempts to isolate such a virus always fail, de-spite huge financial incentives and numerous attempts to do so by an enormous army of scientists dedicated to "HIV," whereas far less interesting viruses are routinely isolated by much smaller, less-funded groups of virus hunters; (2) what is called HIV RNA and DNA comes in many sizes and varieties that always differ from each other (no two are alike, even when abstracted from the same patient ), whereas viral RNA and DNA should be of uniform length and composition; (3) the lethargy that characterizes what is considered "HIV replication" excludes the possibility that replicative mutation can explain the wide HIV genetic variation; and (4) no one has produced a whole "HIV RNA" molecule or a complete "HIV DNA" strip, offering instead as the "HIV genome" cobbled together bits of genetic material.

Papadopulos notes that when "HIV DNA" shows up, it does so in only a tiny fraction of T4 cells. Duesberg's explanation is that this means HIV simply infects too few cells to explain any disease. But if HIV is so lethargic as to infect only a few cells, how can its amazing variability be explained? Papadopulos' hypothesis predicts wide variability: if "HIV DNA" originates from the rearrangement of normal cellular DNA sequences, then each one originates independently and separately in each cell where it is found. Various points of origin would result in a variety of recombination products: DNA strips of varying lengths and composition, and corresponding RNA molecules transcribed from that DNA.

Papadopulos stresses that her argument against the existential hypothesis of HIV does not require that her alternative hypothesis be correct. Since the existence of HIV is not a default hypothesis, we are not obligated to assume that HIV exists in the absence of a better explanation. To the contrary, until unambiguous evidence is provided for HIV--in the form of a proper viral isolate--explanations for the data are open to suggestions. As far as the Australians are concerned, the viral model has been thoroughly examined, and it comes up empty. It's time to propose and study some new ideas.

The Duesberg-Papadopulos dichotomy

Papadopulos' advocacy of a non-viral explanation for microbiological phenomena labeled as "HIV" remarkably resembles Duesberg's advocacy of a non-HIV explanation for pathological phenomena labeled as "AIDS": (1) Duesberg explains that the HIV-AIDS correlation is not as high as it's made out to be; Papadopulos makes the same claim about the HIV protein-DNA/RNA correlation; (2) Duesberg shows that the microbiological data unqualifiedly exclude a role for HIV; Papadopulos shows that the microbiological data unqualifiedly exclude definitive evidence of a virus; (3) Both say we should therefore consider non-viral explanations; and (4) Duesberg says that even if the alternative hypotheses are ultimately falsified, the HIV-AIDS model is not consequently resurrected, because it fails all on its own; Papadopulos says the same thing about the HIV existential model.

The February/March 1997 Continuum carried a second appeal from Duesberg responding to the Papadopulos and Lanka rebuttals. The editors entitled the article, "Near Enough Is Good Enough?" reflecting their sympathy for the non-existentialist position. Duesberg restated his conclusions that rearrangement of normal chromosomal DNA sequences was less likely than the viral explanation, and that the traditional virus isolation requirements advocated by Papadopulos and Lanka were outdated and, in any case, less rigorous than those which he said had been achieved by HIV.

This defense of HIV's existence recalls the arguments used against Duesberg's own proposal that HIV is harmless. Within that discussion, Duesberg shows that HIV fails to meet the traditional and logical standards of microbiology, including Koch's postulates. Advocates of the HIV-AIDS model respond by proclaiming those criteria are outdated, and offer new criteria which accommodate the HIV-AIDS model.

The Australian response is summarized in the title, "Why No Whole Virus?", and reemphasized points made in their previous exposition.

Electron microscopy

More interesting was Lanka's second rebuttal to Duesberg, which included some new insights. Lanka expounded on the implications of a lack of "HIV isolates" despite dogged efforts. This should not be so for a virus that exists. Lanka writes:

It has been long known that what "AIDS" researchers have presented as photos of "HIV" show normal cellular [microvesicles]... As those particles are designed, in contrast to viruses, for cellular use only, they are very unstable when removed from their context, and not able to be isolated and photographed in an isolated state. Viruses are stable because they have to leave cells or even the organism in order to infect other cells or organisms anew. Using centrifugation techniques it is no problem to separate viruses from all contaminating components and in doing so to isolate them--then photograph them, then represent their proteins and genetic substance in a direct way... Genuine viruses are so stable that it is easy... to photograph them directly as three dimensional particles in the [scanning] electron microscope without prior chemical fixation. In contrast [microvesicles] are so unstable they can only be photographed [with a transmission electron microscope, which requires they be] in a chemically fixed state... in very thin sections. All that have been shown to us as [micrographs of] "HIV" are ultrathin sections [that include what are agreed to be] cellular particles. ..

Sure enough, the micrographs of proper viral isolates presented by Lanka in his rejoinder to Steven Harris were photographed with the scanning electron microscope, and thus showed--with high resolution and three-dimensional relief--the outer surfaces of the viruses. In contrast, the purported "HIV" micrograph presented by Harris was photographed by the transmission electron microscope in "ultrathin sections," producing flat, transparent, cross-sectional images with no surfaces and poor resolution. According to Lanka, viruses are hardy enough to be photographed either way, and ought to be, since one reveals the surface in great detail, and the other reveals important cross-sectional information.

But there exists no published scanned micrograph of anything claimed to be "HIV." Since there are billions of dollars and tens of thousands of scientists annually devoted to the study of "HIV," it seems improbable that this could indicate an oversight. More likely the retrovirus-looking objects called "HIV" are, like microvesicles, simply too unstable for scanned electron microscopy and procedures that could otherwise separate them from all other objects into pure samples, which is to say--in Lanka's opinion--they are too unstable to be viruses.

(Instability, by the way, gives the objects labeled "HIV" both the characteristics Papadopulos assigns endogenous retroviruses, the other being non-infectivity in their cell-free form.)

"'HIV' has never been identified as a secure biological entity," he concludes. "The logical explanation given that all the characteristics ascribed to 'HIV' are well-known cellular entities and characteristics, is that 'HIV' never was, and the claim of the existence of 'HIV' is not sustainable."

On hemophilia-AIDS, T4 counts, and African AIDS

Papadopulos' contribution to the AIDS reappraisal movement transcends the discussion of HIV's existence. Remember that she unifies all the proposed causes of AIDS, and even the agents required for "HIV" expression, by a common denominator: they all cause oxidative stress. She also shows that oxidation is a logical source of many diseases, including all that qualify as "AIDS."

In 1995 her team published a lengthy consideration of "AIDS" in hemophiliacs, "Factor VIII, HIV and AIDS: An Analysis of Their Relationship" (Genetica 95: 25-50). To their assertion that factor VIII contaminants cause AIDS conditions in both HIV-positive and -negative hemophiliacs, they also stress a point promoted by no other reappraising scientists: that there is not even a basis for HIV transmission via Factor VIII injections--or any other mechanism, for that matter--since what is called cell-free "HIV" is bare of the surface protein (gp160) supposedly required for infection.

The Australians have also advanced--with Bruce Hedland-Thomas and Barry A. P. Page joining Papadopulos and Causer from the Medical Physics Department at Royal Perth Hospital--another novel hypothesis, this one refuting the role of lost T4 cells in AIDS. In "A Critical Analysis of the HIV-T4-cell-AIDS Hypothesis," they argue that the progressive drop in T4 counts observed in many AIDS patients does not reflect a loss of T4-cells. Rather, it indicates the conversion of many T-cells from producing T4 surface markers to producing T8 markers instead. Thus there is no need to propose a T4-specific factor, such as HIV, to explain AIDS.

Then there is the issue of AIDS in Africa, where the symptoms and proposed causes are often quite different than in the industrialized world. In 1995 the Papadopulos team published "AIDS in Africa: Distinguishing Fact and Fiction" (World Journal of Microbiology and Biotechnology 11: 135-143), co-authored by PhD biologist Harvey Bialy, research editor of Bio/Technology who has spent a great deal of time in Africa. This paper attributes AIDS cases there to the same thing that causes identical symptoms (persistent fever, wasting, and diarrhea) in Africans who test negative: extreme poverty, featuring subsistent diets and rudimentary or nonexistent sanitation.

The paper also explores the implications of the very poor heterosexual transmission rate (one per thousand unprotected contacts with a positive person) assigned to HIV in the face of high fractions of African heterosexuals testing HIV-positive. Either African heterosexuals are much more promiscuous than their American counterparts, or HIV tests are especially problematic in Africa.

The Australians show that problematic testing is the more likely explanation. Malaria, tuberculosis, and other tropical microbes that are widespread in Africa feature proteins that elicit the same antibody response as some of the "HIV proteins." HIV proponents have not accounted for this in any of their experiments. They simply assume that Africans who test positive are indeed infected by HIV, when these tests may instead be indicating very common and conventional infections.

Gordon Stewart joins Papadopulos

"It seems tragic," Duesberg said in one of his Continuum papers, "that over 99% of the AIDS researchers study a virus that does not cause AIDS and that the few who don't are now engaged in a debate over the existence of a virus that doesn't cause AIDS."

Charlie Thomas, the retired biochemistry professor who used to teach at the medical schools at Harvard, John Hopkins, and the University of Michigan, takes a more popular view. "The debate over HIV's existence instigated by the Australians," he has said, "is the only issue of high scientific interest that has emerged from this HIV/AIDS mess."

The "HIV non-existentialists," as Duesberg calls them, acquired an important endorsement this year from the eminent British epidemiologist and physician Gordon Stewart, who is emeritus public health professor at the University of Glasgow in Scotland. Stewart co-authored the Australians' latest paper, "HIV Antibo-dies: Further Questions and a Plea for Clarification" (Current Medical Research and Opinion 13:627-634), which argues that "the evidence for the existence of HIV and its putative role in AIDS must be reappraised."

Voltaire, though, might side with Duesberg on this one. He said, "To not be occupied and to not exist amount to the same thing." And Duesberg and Papadopulos do agree on one thing. There is no HIV occupied with AIDS-causing activities. *

ISOLATION 101: THE BASICS

By Paul Philpott

Reappraising AIDS June, July, Aug. 1997

Isolating a virus

To isolate a virus, scientists take a heterogeneous sample (fluid from a patient or a culture) and add it to a graduated density gel, which they spin in a centrifuge. The contents of the sample settle into separate piles, or bands , at different depths according to their characteristic densities. These bands are called density-purified samples .

Because all microbiological entities have characteristic densities, scientists can obtain density-purified samples that contain only certain viruses, and no other material. There is only one way to confirm this: a photograph with an electron microscope that contains nothing but identical virus-looking objects.

If the micrograph reveals contaminating entities, that means the sample contained some material that had the same density as the virus-looking objects. In that case, scientists would have to add additional steps to the isolation process, ones that purify based on other characteristics--like size, or electrical affinity--until they could produce a sample that contained only the virus-looking objects. However, this usually is not necessary. Density purification typically produces true isolates of virus-looking objects.

If the density, appearance, and size of these objects match those of a previously characterized virus, scientists can label the sample a virus isolate. If not, scientists must subject the sample (actually, a fresh sample, since electron microscopy destroys whatever it photographs) to a battery of tests to prove that the virus-looking objects are viruses.

Proving an isolate consists of viruses

Looking like a virus is just one feature of a virus. To be a virus, virus-looking objects must behave like a virus, and their constituents must relate to each other in special ways. Scientists demonstrate these criteria by adding an isolate of virus-looking objects to a culture of suitable cells. If the isolate consists of viruses, they will infect the cells and multiply to numbers much greater than those present in the original isolate.

Scientists confirm this by attempting to re-isolate the virus-looking objects from the culture after enough time has passed for substantial viral replication to have taken place. The new isolate should form at the same density as the original, and contain objects that look the same as those in the original sample. But the new isolate should consist of a much thicker band, indicating a larger number of viruses.

Scientists also have to examine the constituent molecules of the isolate. Among other things, they have to confirm that the DNA or RNA codes for all the proteins.

This being the case, scientists declare that the objects are indeed viruses, and that these viruses are characterized by a certain size, shape, and appearance, and consisting of a particular number of proteins and genetic molecules of certain molecular weights or base pair lengths.

Isolating viral constituents

To isolate the contents of a virus, scientists must dismantle the viruses into their constituent molecular parts. They do this by adding a special detergent, SDS, to a viral isolate. The isolate will then consist of the individual molecules that compose the viruses. These molecules include proteins that decorate and line the outer membrane envelope, the globs that form the hollow inner core, and the contents of the inner core: enzymes and DNA or RNA.

Next scientists separate these molecular species from each other, using electrophoresis ,, whereby an electric field pulls the molecules through a gel so that they band according to their weights (instead of densities). Some of the bands will contain proteins, and others contain genetic material, either RNA or DNA.

Scientists call an electrophoresed sample a Western blot if they are considering the bands that contain proteins, a Southern blot if they are considering the bands that contain DNA, and a Northern blot if they are considering the bands that contain RNA. (The unusual names are a salute to E. M. Southern, the scientist who devised this process.)

Protein bands and their constituent molecules are named according to the weight (in daltons) of the molecules. The prefix "p" stands for protein , and "gp" stands for glyco-protein (glyco meaning that the protein has some sugar molecules stuck to it). RNA and DNA bands are named according to the number of nucleic acids or base pairs (in kilobases) that make up the constituent RNA or DNA molecules.

Proving a virus causes a disease

If a virus is hardy and abundant enough to cause a disease, scientists should have no trouble isolating it from the cell-free fluids of affected tissue. This is exactly what scientists must do to convict a virus of causing a disease. They select a group of people who have the disease, and try isolating the virus from the patients' plasma (cell-free blood), or other fluids, depending on the disease. If they fail to isolate the virus from some of the patients, then they must absolve that virus of responsibility for any progressive disease in those individuals, and conclude those people are sick for some other reason.

But what about patients who do present isolatable amounts of the virus? Is that virus responsible for their conditions? Or is the virus an innocent bystander? After all, most viruses cause no disease.

Only microbiological experimentation can establish the culpability or innocence of a virus isolated from the fluid of diseased tissue. To do this, scientists prepare cultures of healthy, uninfected cells of the type damaged or destroyed in the patient. They add viral isolates and watch to see if this effects the culture cells in ways that can explain the disease.

Understanding viral tests

Although isolation is the only direct evidence of a virus, cost and time considerations make it impractical for clinicians. Among other things, for example, it requires confirmation by an electron microscope.

Viral tests, on the other hand, are much simpler. Most require clinicians to just add patient fluids (usually plasma, depending on the virus in question) to the tests and look for reactions to take place.

Scientists construct these tests using components abstracted from viral isolates. Some of the proteins from viral isolates, for example, will react with antibodies secreted into plasma by the immune systems of patients infected by the virus. Antibody tests consist of those proteins. Genetic tests consist of probes made from the DNA or RNA contained in viral isolates. The probes react with viral RNA or DNA in patient fluids.

When constructed and validated properly, and used under the proper circumstances, viral tests can be nearly as accurate and reliable as viral isolation itself. The need for proper test validation and result interpretation stems from the fact that the reactions upon which they depend (antibody-antigen interactions, and genetic probing) are not perfectly specific. Antibodies against one viral protein can react with a similar protein from other microbes, or even some non-microbial proteins. Similar proteins mean similar gene sequences, so genetic tests are less than perfectly specific as well.

Furthermore, even when tests react with their intended viral entities, this doesn't necessarily mean the patient has an active infection, the only sort that can cause disease. Antibodies, for example, can circulate for years—even a lifetime—after the host immune system has suppressed a viral infection to permanent and harmless latency, or even eliminated it entirely. Viral genetic material can also persist in the plasma and other fluids during viral latency.

Therefore, viral tests cannot absolutely and unambiguously identify an actively infected person. Only isolation of objects that possess the appearance and density of the virus in question can do that. So viral tests must not only be constructed from viral isolates, they must also be validated against their ability to predict patients from whom scientists can obtain viral isolates.

Validation studies tend to show that positive test results are highly accurate for patients who express the symptoms that the virus has been proven to cause. On the other hand, positive results are usually very inaccurate for people who have no symptoms. In other words, the virus can be isolated from some very large fraction of positive testing people who express the associated symptoms, but only from a small fraction of positive testing people who express no symptoms. Thus positive tests in healthy people usually don't indicate active infections.

Antigens and antibodies

One measure of the immune system's response to a substantial viral infection is the production by B-cells of proteins called antibodies. Antibodies latch onto and neutralize other proteins.

Proteins that elicit an immune response are called antigens. Viral antigens tend to be those proteins that compose the inner core, and those that decorate or line the outer membrane envelope. These are the proteins that the immune system "sees," whereas proteins inside the core—the viral enzymes—are shielded from immune surveillance. The immune system does not respond to non-protein molecules, like RNA and DNA.

Western blot antibody tests and ELISAs

Scientists construct Western blot antibody tests by transferring to paper some of the protein bands from a Western blot gel. These bands will react when exposed to fluid that contains antibodies against the proteins in the bands.

Another test called the ELISA consists of viral isolates for which the constituent molecules have been broken apart from each other, but have not been separated from each other by electrophoresis. ("ELISA" stands for Enzyme-Linked Immuno-Sorbent Assay, which describes how positive reactions are demonstrated chemically.) This makes ELISAs easier and cheaper to make than Western blot tests.

But ELISAs are not as accurate. People test ELISA-positive if their plasma contains antibodies against just one of the viral proteins, whereas Western blot tests consist of the proteins separated into different bands, so clinicians can see exactly which proteins react with a person's plasma.

ELISAs are usually used as screening tests. Since people who test ELISA-negative have antibodies against none of the viral proteins, negative ELISAs just as accurately identify uninfected people as do Western blot tests showing no reactive bands.

But positive ELISAs are not as good as positive Western blots at identifying people with active infections. This is because there is no such thing as a specific antibody. Antibodies against a certain viral protein may react also with proteins of another virus, or even non-viral proteins. So positivity for antibodies against viral proteins is not unambiguous evidence that a person has been exposed before to a particular virus.

However, people positive for antibodies against all the antigens of a particular virus are much more likely to have been exposed to that virus than someone positive for antibodies against only one or a few of the antigens. Yet each receives the same positive ELISA. Only a Western blot can distinguish these people. Proper validation studies show higher accuracies (fraction of positive subjects from which viral isolates can be obtained) for positive Western blot tests than for positive ELISAs. But since ELISAs are cheaper, Western blot tests are usually reserved for people who first test ELISA-positive.

Northern and Southern blot tests

Southern and Northern blots from viral isolates represent pure samples of viral DNA or RNA. Scientists use the material in these samples to produce viral tests that react with viral RNA or DNA in patient fluids. To do this, they construct small DNA or RNA molecules, called probes, that complement segments of the viral DNA or RNA. To test patients, clinicians make Northern or Southern blots from patient fluid (usually plasma, depending on the virus in question) that has been treated so that any constituent viruses will be broken apart, exposing the genetic material inside.

If there is lots of virus in the plasma, a distinct band will appear in the gel at the location characteristic of the genetic material of the virus in question. Adding the probes will confirm that such a band consists of viral DNA or RNA. If only a small amount of virus exists in the plasma, the genetic material settling at the characteristic location in the gel will become detectable only after the probes are added.

Antigen tests

During the early course of a substantial viral infection, the plasma contains lots of virus, and consequently lots of viral antigens, but very few antibodies against these antigens. This is because the immune response has not yet caught up with the viral activity.

Sometimes there is not even enough antibody to cause a reaction with ELISA or Western blot antibody tests, which contain the antigens. So scientists have developed tests that contain antibodies against viral antigens. These tests react with patient plasma that contains viral proteins. Antigen tests, then, are the inverse of antibody tests.

Viral load tests

Patients rarely ever have enough "HIV RNA" to yield a detectable signal on Northern blots of fresh patient serum. Thus the necessity to invent "viral load" testing, which employs the polymerase chain reaction, PCR. PCR generates millions of RNA or DNA copies out of an original indetectably few molecules.

AIDS reappraisers consider these tests invalid. The concentrations of HIV RNA these assays usually indicate—hundreds of thousands per ml of plasma—would easily show up on Northern blot tests. But it doesn't show up at all.

Active vs. inactive viral infections

Cells with inactive, or dormant, infections have inside them viral DNA molecules, called proviruses, that are asleep. Sleeping proviruses produce no virus, and thus can cause no disease, since viral replication is what destroys or damages cells in the course of a viral disease.

Viral DNA goes to sleep when the host immune system gains the upper hand. Among the anti-viral molecules secreted by immune cells are substances that put viral DNA to sleep. When immunity is suppressed, the plasma levels of these substances diminish, and sleeping viral DNA awakens to start producing new viruses, which show up in the plasma.

Since cell cultures contain no immune systems, they contain none of these anti-viral substances. That makes them ideal nurseries for viruses. When cultures are made from cells containing dormant proviruses, the proviruses have their ideal circumstance to spring back to life and generate a maximum amount of new virus. Some proviruses awaken from dormancy only when stimulated by agents that promote viral activity. These sorts of viruses make very poor candidates for disease causation, for obvious reasons.

The culture skinny

Viruses replicate in two sorts of cells, in vivo (those inside living organisms, such as people), and in vitro (those maintained in laboratory culture dishes). Virus isolation from human plasma demonstrates in vivo viral activity, and isolation from culture fluids demonstrates in vitro viral activity.

However, isolation from the fluids of a culture composed of donor cells can not demonstrate that the donor harbors an active infection. It only demonstrates that the donor cells contain proviruses that are active under culture conditions. Transferring cells from a living organism to a lab dish can permit sleeping proviruses to awaken. Only examination of uncultured tissue fluids can diagnose viral disease.

RNA and DNA viruses

Viruses carry in their core only one sort of genetic material, either RNA or DNA molecules. These molecules are called proviruses when they reside inside a host cell, outside the viral core. Proviruses direct the production of all viral components, even replication of themselves, in the manufacture of new viruses.

Except for retroviruses, viruses that carry RNA are always active, but viruses that carry DNA can be active or inactive.

This is because RNA constantly produces proteins when it is in contact with amino acids (protein building blocks) and ribosomes (enzymes that translate RNA molecules into corresponding protein molecules). In the viral core, viral RNA has no contact with amino acids or ribosomes. But inside a host cell, viral RNA has all the material it needs to produce new viral proteins.

DNA, on the other hand, can not be directly translated into proteins. First it must be transcribed into RNA by an enzyme called transcriptase. But DNA has the ability to regulate its own transcription. So DNA can be active or inactive, whereas RNA can only be active.

Enzymes called reverse transcriptase will reverse transcribe retroviral RNA into corresponding DNA molecules. Consequently, retroviruses share with DNA viruses the ability to be either active or inactive.

Virus counting

How many viruses do infected people have circulating in their blood? There is only one way to answer this question definitively, and it of course involves preparing an isolate from patient plasma, and counting the viruses in the isolate.

Scientists start by obtaining from the patient a fluid sample, which they serial dilute,. Serial dilution results in one undiluted sample, and several others of equal volume diluted by varying amounts. From each sample scientists prepare a viral isolate, which they view on a standard grid using an electron microscope. If the patient has a high viral concentration, the undiluted sample will contain too many to count, since the viruses will be stacked on top of each other, and overlap.

The viruses in one of the diluted isolates will be spread out enough so they can be counted accurately against the grid, which represents some fraction of the area occupied by the sample. By multiplying factors that account for the gridding and diluting of the sample, counting the number of viruses in the grid will yield the number of viruses present in the undiluted sample. Dividing this number by the pre-diluted volume yields the viral concentration (in particles per milliliter) in the patient's plasma.

This of course is too expensive and complicated for the clinical setting. So scientists can calibrate some of the viral tests to approximate viral concentrations. For example, the thickness and staining intensity of Northern and Southern blot bands are directly proportional to viral concentration. So is the staining intensity of antigen tests. So by examining these test results for patients who have had their viral concentrations established, scientists can derive numbers that convert band thickness or staining intensity into viral concentration.

Tissue Culture Infectious Doses (TCID)

One of the ways that clinicians can characterize a viral infection is to approximate the plasma concentrations of Tissue Culture Infectious Doses, or TCIDs. One TCID is the minimum amount of virus required to produce viral activity in a standard culture of stock laboratory cells. Scientist determine viral activity by obtaining viral isolates, or by observing phenomena previously shown in isolation studies to be viral, such as the appearance of certain proteins.

To determine TCID concentrations, scientists take plasma from a patient and serial dilute it. Serial dilution involves producing from an original sample a sequence of samples, each of the same volume, but each one ten times more diluted than the previous. Thus going down the line from the original sample to the last, each will contain in relation to the previous one, a tenth of the plasma—and a tenth of the viruses—contained in the original sample.

Each sample is added to a separate standard culture. If the undiluted sample causes no replication, then neither will any of the diluted samples, and the plasma contains no TCIDs. If the undiluted sample does cause replication, then it contains at least one TCID. If the first diluted sample causes replication, then the undiluted sample contained at least ten TCIDs. If the second diluted sample produces no replication, then the original sample contained at least 10, but fewer than 100, TCIDs. This range can be narrowed down by now using a dilution factor smaller than ten.

Because the usual dilution factor is ten, this process is called titration, and the term TCID can be substituted with the term titer (or titre). In the above example, scientists would say that the original sample had 10 TCIDs, or a viral titer of ten. By dividing this figure by the volume of the original sample, they can calculate the plasma concentration for the patient, in TCIDs per milliliter.

Validation studies can correlate TCID concentrations with actual viral concentration. However, this usually is not done, because TCID values provide more important information than viral concentrations. Remember, only infectious viruses can cause disease, and only if they are present at concentrations great enough to cause a productive infection. So it is more important to know the concentration of TCIDs than the concentration of actual viruses. *

REMARKS ON METHODS FOR RETROVIRAL ISOLATION

By Etienne de Harven

Continuum Spring

Dr. Etienne de Harven is emeritus Professor of Pathology, University of Toronto. He worked in electron microscopy (EM) primarily on the ultrastructure of retroviruses throughout his professional career of 25 years at the Sloan Kettering Institute in New York and 13 years at the University of Toronto. In 1956 he was the first to report on the EM of the Friend virus in murine (mouse) leukemia, and in 1960, to coin the word "budding" to describe steps of virus assembly on cell surfaces. He will deliver a speech at the 12th World AIDS Conference in Geneva (June 28-July 3) at the session "HIV-testing: Open Questions about Specificity".

The most impressive developments of molecular genetics over the past 20 years do not make Robert Koch's postulates obsolete. The first of these postulates indicates that to be considered as pathogenic, a microorganism should be isolated in every single case of the disease. Still, according to E. Papadopulos et all and S. Lanka (2) isolation of HIV from fresh plasma of AIDS patients has never been achieved under any circumstances. Moreover, and most surprisingly, the "efficiency" of current antiviral therapeutic protocols (AZT tri-therapy) is being measured by determining "viral load" in the plasma of treated patients. "Viral load" implies viremia I.e. the presence of circulating viral particles in the peripheral blood. The virus incriminated being allegedly a retrovirus, this would have been the time to remember that the morphology of such viruses in several animal experimental tumors and leukemias had been extensively characterised by transmission electron microscopy (EM) over the past 40 years, the viral particles having a characteristic ultrastructure and a diameter ranging between 100 and 120 um. Some of them had been studied by methods of high resolution transmission electron microscopy.(3) In the 1960s, transmission electron microscopy was by far the best available method to identify viruses within or around diseased cells. Consequently, many cancer research centers all around the world, started to compete for the best equipment and training in EM, aiming at the demonstration in human malignancies of viruses similar to those which had just been recognized as significantly associated with tumors and leukemias of several laboratory animals. This approach to cancer research appeared highly justified when Lwoff, Horne and Tournier proposed to classify all viruses primarily on the basis of their morphological features demonstrated by electron microscopy.(4) Identification of viruses by EM in leukemic animal tissues became unambiguous when steps in virus assembly, i.e. the 'budding' of complete virions from the surface of the infected cells, were described.(5) In spite of considerable efforts, the search for similar, typical viruses in human malignancies remained entirely negative. Pleomorphic membranous microvesicles, approaching viral size, and frequently described in the literature on human malignancies as "virus-like particles" were without any pathogenic significance. As stated in 1965, typical RNA tumor viruses have never been observed in association with human neoplasia.(6)

Concentrations of retroviruses from murine and avian leukemic tissue homogenates were reproducibly achieved permitting titration of infectivity into receptive laboratory animals. This was not, however, an easy approach to the problem of virus purification, large amounts of microvesicles and cell debris being usually present. As far as virus purification was concerned, it soon became evident that when viremia is present, blood plasma was far better than tissue homogenates for efficient virus isolation and purification.

In the case of RNA tumor viruses, now called retroviruses, the demonstration of viremia in the blood plasma of experimental leukemic animals (chickens and mice) was published more than 35 years ago. A most efficient purification method including ultrafiltration and ultracentrifugation of a 1/1 dilution of plasma in heparinized Ringer's solution, allowed me to demonstrate packed retroviruses by transmission electron microscopy (7) in thin sections of pellets obtained by high speed centrifugation of the purified virus, quite clearly establishing that the amount of contaminating cell debris was remarkably small, a conclusion which could never have been reached by using the negative staining EM method. Using this simple ultrafiltration procedure, virions were never exposed to hypertonic shock. However, sedimentation in sucrose density gradients, at the density of 1.16 gm/ml, soon became the most popular method for retrovirus purification.(8) Interestingly, it was very well known by electron microscopists in the 1960s, that sharp bands sedimenting at the density of 1.16 frequently contained large amounts of microvesicles and cell debris of non-viral nature. These debris just happened to sediment in sucrose gradients at a density very similar to that of retroviruses clearly indicating that finding a "sharp band" at the density of 1.16 gm/ml was of little significance and was certainly far from any demonstration of retroviruses isolation.

But this conclusion was based on EM findings, and around 1970 the faith in retroviral oncology was assuming quasi-religious proportions! If EM cannot demonstrate viruses in the 1.16 bands, let us forget about EM and rely on other "markers"!

When around 1980, R. Gallo and his followers attempted to demonstrate that certain retroviruses can be suspected of representing; human pathogens, to the best of my bibliographical recollection, electron microscopy was never used to demonstrate directly viremia in the studied patients. Why? Most probably, EM results were negative and swiftly ignored! But over-enthusiastic retrovirologists continued to rely on the identification of so-called "viral markers", attempting to salvage their hypothesis.

When retrovirus particles are legion, the study of molecular markers can be useful, and provide an approach to quantification probably better than direct particle counting under the EM (which I always found very difficult). But when, using EM, retrovirus particles are absent relying exclusively on 'markers' is a methodological nonsense. 'Markers' of what?

Nevertheless, for the past ten years, HIV research and clinical therapeutic trials have been primarily based on the study of several HIV "markers".

First the antibody. Elisa, then Western Blot tests were hastily developed (at sizable financial profit eagerly split between the Pasteur Institute and the US). "Seropositivity" became synonymous with the disease itself, plunging an entire generation into behavioral panic, and exposing hundreds of thousands of people to 'preventive' antiviral AZT therapy which actually hastened the appearance of severe or lethal immunodeficiency syndrome. Appropriate controls were apparently never carried out or were never published. Still, back in 1993 it became clear that the so-called HIV antibody tests badly lacked specificity, (9) cross-reactivity being observed with patients suffering from a long list of pathological conditions including malaria, leprosy, auto-immune diseases and many more.

Secondly, 'viral proteins'. Several proteins have been identified as 'HIV markers', most frequently because they were identified in a variety of 1.16 bands. The case of the p24 "viral" antigen is a significant example and its lack of viral specificity has been well documented.(10)

Third, reverse transcription. If reverse transcriptase activity were a unique feature of retroviruses, it could have been an interesting molecular marker. Unfortunately, it has been shown that reverse transcriptase is found in the uninfected cells of yeasts, insects and mammals (11) and "has nothing to do with retroviruses as such" as well referenced in a recent report from S. Lanka. Moreover, K. Mullis himself does not support the use - to amplify and quantify the "HIV genome" - which is being made of the PCR methodology he developed, which is the current method of "measuring the viral load" in AIDS patients.

More disturbing is the fact that some 'markers' are searched for in the 1.16 gradient sedimenting material which is the density where intact virions are expected to be found, but not their molecular fragments. If lysed retrovirus particles released molecular markers, the 1.16 samples should at least initially allow investigators to demonstrate virus particles by EM. They don't. however after 15 years of most intensive HIV research, two independent groups finally decided to explore by electron microscopy the ultrastructural features of the material sedimenting at the 1.16 density. Working on "HIV-1 infected T-cell" cultures supernatants, both groups found that it contains primarily cellular debris and cell membrane vesicles which could definitely not be identified with HIV particles and rare "virus-like" particles.(12, 13) Still, this is the type of sample in which "viral markers" are currently identified and used to measure the effects of anti-viral drugs in current clinical trials.

In conclusion, and after extensive reviewing of the current AIDS research literature, the following statement appears inescapable: neither electron microscopy nor molecular markers have so far permitted a scientifically sound demonstration of retrovirus isolation directly from AIDS patients. This conclusion fully confiens the recent reports published in Continuum by E. Papadopulos and by S. Lanka.

Harvey Bialy, editor of the journal Bio/Technology has stated that (14) "A powerful hypothesis has to explain and predict. What kind of scientist continues to support a hypothesis that fails to explain and fails to predict?" The HIV/AIDS hypothesis fails to explain the considerable drop of T4 circulating lymphocytes in AIDS patients. It predicted a dramatic AIDS epidemic which was never observed (unless we accent the CDC's most surprising redefinition of AIDS as including some 31 "AIDS defining illnesses"!).

Obviously, the HIV/AIDS hypothesis has to be scientifically reappraised.(15) And, most urgently, the funding for AIDS research should no longer be restricted to laboratories working on an hypothesis which has never been proven. *

References

1. Papadopulos-Eleopulos E, Turner VF, Papadimitriou JM, Causer D, Hedland-Thomas 13, Page B, 1994. A critical analysis of the HIV-T4-AIDS hypothesis. Genetica 95:5-24

2. Lanka, Stefan, 1994. Fehldiagnose AIDS? Wechselwirkung, Aachen, December, 48-53.

3 de Harven, E, 1974. remarks on the ultrastructure of type A, B and C virus particles. Advances in Virus Research 19: 221-264. Academic Press, Inc., publ., New York.

4. Lwoff A, Horne R, Tournier P, 1962. Cold Spring Harbour Symposium on Quantitative Biology 27:51.

5 de Harven E, and Friend C, 1960. Further electron microscope studies of a mouse leukemia induced by cell-free filtrates. J. Biophysic. and Biochem. Cytol., vol 7, 747-752. Rockefeller University Press, New York

6. de Harven E, 1965. Remarks on viruses, leukemia and electron microscopy. In Methodological approaches to the study of leukemias. V Defendi, edit., The Wistar Institute Press publ, Philadelphia, pp147-156

7. de Harven E, 1965. Viremia in Friend leukemia: the electron microscope approach to the problem Pathologie-Biologie,13:125-134 de Harven E, 1998. Pioneer deplores "HIV". Continuum vol 5, page 24

8. Sinoussi F, Mendiola L, Chermann JC, 1973. Purification and partial differentiation of the particles of murine sarcoma virus (M.MSV) according to their sedimentation rates in sucrose density gradients. Spectra 4:237-24

9. Papadopulos-Eleopulos E, Turner VF and Papadimitnou JM, 1993. Is a positive Western Blot proof of HIV infection? Bio/Technology 11:696-707

10. Todak C, Klein E, Lange M et al., 1991. A clinical appraisal of the p24 antigen test. International Conference on AIDS, vol 1, Florence, Italy

11. Varmus H, 1987. Reverse transcription Sci. Am. 257:48-54

12. Gluschankof P. Mondor I, Gelderblom HR, and Sattentau QJ, 1997. Cell Membrane vesicles are a major contaminant of gradient-ennched human immunodeficiency virus type-l preparations. Virology 230:125-133

13. Bess JW Jr., Gorelick WJ, Bosche WJ, Henderson LE, and Arthur LO, 1997. Microvesicles are a source of contaminating cellular proteins found in purified HIV-I preparations. Virology 230:134-144

14. Farber, C, 1992. Fatal distraction, Spin Magazine, June 1992

15. Philpott P, 1997. The isolation question. Reappraising AIDS, vol 5 number 6, 1-12

Author's address:
Dr Etienne de Harven
le Mas Pitou,
2879 Route de Grasse
06530 Saint Cezaire sur Siagne, France

WHAT IS THE EVIDENCE FOR THE EXISTENCE OF HIV?

By Valendar Turner

Department of Emergency Medicine, Royal Perth
Hospital, Perth, Western Australia

The real purpose of scientific method is to make sure Nature hasn't misled you into thinking something you don't actually know... One logical slip and an entire scientific edifice comes tumbling down. One false deduction about the machine and you can get hung up indefinitely.

Robert Pirsig, Zen and the Art of Motorcycle Maintenance

Does the currently available evidence prove beyond reasonable doubt that a unique, exogenously acquired retrovirus has been isolated from the tissues of AIDS patients? Perhaps. Perhaps not. This is what I invite you to judge. And in case you are inclined to be assaulted by the opinions of overwhelming majorities, you may take comfort from a most venerated, international scientist who said, "In Science the authority embodied in the opinion of thousands is not worth a spark of reason in one man." I shall reward you with his identity at the end of this talk.

A virus is two things. Number one: It's a microscopic particle of certain size and form. Number two, such particles generate identical progeny by parasitising chemical constituents and energy from a living cell. This is what is actually meant by the term infectious. It is this attribute which justifies a particle being called a virus. This is the property which prevents our calling every particle we see a virus. By definition, a retroviral particle is spherical in shape and has a diameter of 100-120 Nm. On the outside is a shell studded with outwardly projecting knobs, knobs obligatory to latch on to and infect new cells. On the inside there is a core containing RNA as well as some proteins, one of which is an enzyme called reverse transcriptase. The latter gives retroviruses their name and its function is to catalyse the transcription of viral RNA into DNA, that is, to copy information contained in RNA in a direction opposite the customary direction, DNA to RNA. According to virologists, it is the DNA copy of the RNA blueprint, not the original RNA, which hibernates inside the cell nucleus awaiting an opportune time to orchestrate the production of new viruses.

To analyse their constituents and to prove they are truly viruses, retroviral-like particles must first be purified. This is done by a process called density gradient ultracentrifugation, something that may sound complicated but which isn't. A test tube containing a solution of sucrose, ordinary table sugar, is prepared light at the top, but gradually becoming heavier towards the bottom. A drop of fluid from a cell culture is gently placed on top and the test-tube is centrifuged for several hours at extremely high speeds. This generates forces many thousands of times that of gravity and any tiny particles present are gradually forced through the sugar solution until they reach a point where their buoyancy prevents them penetrating further. For retroviral particles, this occurs where the density reaches 1.16 gm/ml, the point where the particles concentrate or, to use virological jargon, band. The 1.16 band can then be selectively extracted and photographed with an electron microscope. So, to prove the existence of a retrovirus one is obliged to:

1. Culture putatively infected cells.

2. Purify a sample in a sucrose density gradient.

3. Photograph the 1.16 band proving there are particles of the right size and form, and there is no other material.

4. Extract and analyse the constituents of the particles and prove they contain reverse transcriptase by showing they can make DNA from RNA.

5. Culture purified particles with virgin cells demonstrating that a new set of particles appears with the same morphology and constituents as the originals.

Now I am going to discuss some of the data from four papers published in Science in May 1984 by Dr. Robert Gallo and his colleagues from the US National Cancer Institute. These papers do not describe the original discovery of what the overwhelming majority regard as HIV, that distinction falls a year earlier to Professor Luc Montagnier and his colleagues from the Pasteur Institute from where, it is important to say, samples were sent to the Gallo laboratory and which later caused Gallo and his colleagues, as well as the US government, quite a number of problems. Neither are the Gallo papers the last word on HIV isolation but there is no doubt they are most important because it was they that led to the famous Washington press conference of April the 23rd 1984, two weeks prior to publication, at which an anxious, waiting world was told that the cause of AIDS had been identified. In fact, as one scrutinises the vast AIDS literature, it is fair to say that of all the papers published on HIV isolation, including the very latest, the Gallo papers are the most rigorous by far. The problem is, are they rigorous enough?

The first paper begins with cultures made of T-lymphocyte cells from AIDS patients. These cells were chosen because, included amongst their numbers, are the putatively infected cells, a subgroup known as T4 lymphocytes. It is these that are often diminished in AIDS, the hypothesis being that the yet to be discovered retrovirus was infecting and killing them. After an unspecified time, concentrated fluids from these T-cell cultures were subcultured with cells of a stock, leukaemic T-cell line known as HT. In these secondary cultures the Gallo team reported particles in electron microscopic examination of gross, unrefined culture fluids and measured reverse transcriptase activity in both these and banded specimens but without evidence that retroviral- like particles or indeed any particles were present at 1.16 gm/ml. They also reported reactions were seen between culture proteins and some antibodies present in human and animal sera. From these data, the Gallo team claimed to have isolated a new retrovirus, HIV, as well as inducing it to grow in the HT cell line in large enough quantities for use in analysis and diagnosis. In a subsequent third paper, from banded culture fluids obtained from a disrupted HT cell clone, two proteins, and for no other reason than they reacted with antibodies present in human AIDS sera, were deemed to be the HIV proteins. Subsequent papers, published after the Gallo four, using the same logic, increased the number of such proteins to about ten.

Reading these data it is obvious that Gallo and his colleagues had abandoned the traditional method of retrovirus isolation. This is enigmatic when one realises that, in 1976, Gallo himself had stressed that the detection of particles and reverse transcriptase, even reverse transcriptase inside particles, are not proof of the existence of retrovirus because, no matter how remarkably such particles may resemble retrovirus, many such particles are not viruses because they totally lack the ability to replicate (Gallo et al., 1976). You must appreciate the magnitude of the particle problem. Cell cultures contain many and many kinds of particles, some viral-like and some not. The viral-like include retroviral-like. In the 1970s, retroviral-like particles were frequently observed in human leukaemia tissues (Gallo et al., 1976), cultures of embryonic tissues, and "in the majority, if not all, human placentas." (Panem, 1979) One genus of retroviral-like particles, the type-C particle and the one into which Gallo classified his newly discovered retrovirus HIV, is found in "fish, snakes, worms, pheasant, quail, partridge, turkey, tree mouse and agouti" (Grafe, 1991) as well as in "tapeworms, insects...and mammals." (Frank, 1987) This being the case, there seems to be no way of avoiding the rules developed over the decades of research into animal retroviruses, rules that enabled a scientists to sort out this clutter. And there are two more complicating factors. The first is that reverse transcription is not only a property of retroviruses. Normal cells contain enzymes which reverse transcribe RNA and so does hepatitis B virus, a virus that infects T-cells as well as liver cells and is present in a considerable number of AIDS patients. The second is the choice of the HT cell line. It was long known that leukaemic cells theselves can reverse transcribe and, strange as it may seem, although Dr. Gallo was about to look for reverse transcription as a sign of a new retrovirus, the HT cell line originated from a patient who, according to Dr. Gallo, had a disease caused by a retrovirus he discovered called HTLV-I. In fact, in 1983, Gallo reported that the HT parental cellline contained HTLV-I genetic sequences. On this basis alone one would expect to find evidence of reverse transcription in the HT cell line. Given all these data, one would imagine it was impossible for the Gallo team to abandon the need to follow the traditional method and isolate and characterise infectious particles, but abandon it they did. By what reasoning then did the Gallo team claim to have proven the existence of a new retrovirus from AIDS patients?

For their 1984 papers, they reiterated the limitations of particles and reverse transcriptase and made three assumptions which, taken together, constitute a precept known as specific reactivity. (Gallo et al., 1986) The first assumption was that AIDS patients are infected with a replicating retroviral particle, a virus which could be grown in cell cultures to yield unique, virus-specific proteins. Second, being foreign, the virus would stimulate the production of a number of distinctive antibodies directed against the viral proteins. Third, the proteins and the antibodies react specifically, that is, only with each other and with no other agent. Let us take a very careful look at this paradigm. First, when the Gallo team began their experiments, the existence of specific viral proteins as constituents of a replicating viral particle which could infect humans was entirely hypothesis, not fact. Second, antibodies and proteins are not monogamous, even the purest of each take on other partners. Third, even if they were monogamous, we know that AIDS patients contain antibodies to many different agents, many with which they are infected, for example hepatitis B and cytomegalic inclusion viruses, mycoplasma, fungi and mycobacteria. Unless Gallo further hypothesised that all these agents or parts of them, or their respective antibodies, disappear from cultures or sera, when blood from an AIDS patient is mixed with cell cultures of the same or another AIDS patient, how can anyone tell what is reacting with what, let alone define precisely where each of the reactants originated? As far as the reactions are concerned, it's no different from mixing up milk from six species of animals, adding a mixture of a dozen different acids and claiming to know which acid is curdling which milk. So, although the term specific HIV proteins conjures up visions of proteins being extracted from retroviral-like particles proven to be a unique virus, this is not how it was done. It was done by breaking up cells of the HT cell line, not a virus particle, and observing unknown proteins reacting with unknown antibodies. From these data both the proteins and the antibodies were deemed viral, and not just any virus, but HIV. That's all. Logic or magic? And as an aside, similar to the proteins, the origin of what is called the HIV genome, the HIV RNA, is also based on circumstance, not on purification and dissection of particles proven to be infectious. The Gallo team may have claimed isolation of a new retrovirus but what they actually did was weave a nexus between reverse transcriptase, particles, and certain proteins under the dubious imprimatur of specific reactivity. Is this virus isolation? Is this even virus detection?

There are also a number of unsolved mysteries in the Gallo papers.

Mystery number one:

Reading the first paper one gets the impression that the HT cell line was cultured with individual AIDS patient cultures. However, the National Institutes of Health enquiry instigated after allegations of misappropriation of the French specimens found that the HT cell line was cultured with concentrated fluids pooled initially from individual cultures of three patients and ultimately from the individual cultures of ten patients. (Maddox, 1992) In evidence given to the enquiry, the reason given was because none of the individual cultures "was producing high concentrations of reverse transcriptase." That means not enough to convince the Gallo team of scientists or anybody else there actually was a virus in any of the individual specimens in the first place. The fact that pooled specimens produced reverse transcription is not proof of a retrovirus. The conditions may have merely changed in favour of the action of one of the cellular enzymes that performs the same trick. Or it could have been due to the HT cell line, unaided or at the behest of its HTLV-I retrovirus. The Gallo investigation found the pooling of specimens "of dubious scientific rigor." One scientist described the procedure as "really crazy." In essence, it is no different from investigating an outbreak of pneumonia by having all patients spit in separate pots and, when nothing turns up, getting them all to spit in the same pot.

Mystery number two:

The method of specific reactivity required a source of antibodies to the putative viral proteins. The logical place to obtain these was from AIDS patients -- after all, that is what the hypothesis required. The antibodies reported in the first paper appeared from two sources, a haemophiliac patient known as E.T. who had pre-AIDS and rabbits. Yes, rabbits. What precisely constituted E.T's pre-AIDS we are not told, but pre-AIDS is often generalised enlargement of the lymph nodes, a condition not invariably followed by AIDS and which is not AIDS. Thus, according to the paradigm of specific reactivity, we cannot be sure that E.T. actually had the right kind of antibodies. Rabbits do not develop AIDS and if specific antibodies to a retrovirus were to exist, they could only be produced by immunising rabbits with pure virus or, as the first Gallo group paper reported, from rabbits infected repeatedly with disrupted HIV. I hope you are beginning to see the problem. To make antibodies just to HIV, one has to inject rabbits with pure HIV. Pure virus means isolated virus and if rabbits were injected with pure virus, why should it be necessary to produce antibodies to define the isolation of virus that had already been isolated?

Mystery number three:

In the second paper, the Gallo team attempted what they called HIV isolation from 72 AIDS patients. Again, they cultured cells and detected particles and reverse transcriptase in unrefined culture fluids, and observed some protein/antibody reactions, but also added a fourth category, transmission, by which was meant finding particles or reverse transcriptase in bone marrow and other cells cultured with fluids, but not purified banded, photographed fluids, from one of the 72 starting cultures. What is enigmatic about the second paper is that HIV isolation was defined merely as detecting at least two of any of these four phenomena. The same criticism applies as in the first paper. Nothing was isolated and detection of unspecific phenomena is not surrogate isolation of a retrovirus. Even it were, this peculiar definition leads to some rather bizarre possibilities, for example, instances of virus isolation without the need to see particles or measure reverse transcriptase, for a retrovirus about as convincing as trying to sell a car without a body and an engine. Even so, loose as these criteria were, isolation was successful in only 26 of the 72 patients, that is, in only 36%. And, in case you think things have improved, there is a recent, international cooperative study reported by the World Health Organisation. In this study, by HIV isolation was meant detection of a single protein, p24, in culture fluids using a single antibody. Not only is p24 not specific for HIV (Agbalika et al., 1992; Mortimer et al., 1992), but from 224 HIV positive individuals, the success rate was a mere 37%, not significantly better than Gallo's figures a decade earlier. (WHO, 1994)

Even if the Gallo team had proved the existence of a new retrovirus, on what basis did they claim it was the cause of AIDS? Even if virus had been isolated from all patients and all patients had antibodies, which they didn't because in the fourth paper, the data showed only 88% of AIDS patients had antibodies (and on a single ELISA test which no one now regards as specific), is this sufficient proof that HIV causes AIDS? If the bank manager and his constant, faithful offsider are present at the bank robbery, is this proof that the manager robbed the bank? The Gallo papers provide no evidence whatsoever that HIV kills T4-cells or that low numbers of T4-cells is necessary and sufficient for the development of the AIDS infections and cancers and, I might add, there is still no such evidence. (Papadopulos-Eleopulos et al., 1994)

Let me finish by summarising the problem. The method of retrovirus isolation presented at the beginning flows logically from the definition of a virus. It is model of intelligibility, it is the only method, and was used for decades of research into animal retroviruses. (Sinoussi et al., 1973; Toplin, 1973) The problem is that, to date, nobody in the world has reported use of this method in AIDS patients. Without it, for example, how can one resolve the dilemma imposed by the numerous particles of stunning morphological variability present in cell cultures of AIDS patients? Even so, although some particles are the right diameter, there are no particles with the right diameter AND the projecting knobs, both integral to the definition of a retroviral particle and the latter essential to infect new cells. (Gelderblom et al., 1988; Layne et al., 1992; Levy, 1996) Yet, as I speak, there is still not even one published electron micrograph from a density gradient to tell us which, if any constituents of this zoo of particles, presents itself to be proved an infectious retrovirus. Perhaps, if someone were to look, there might not be any. Reading the literature, it is obvious that scientists everywhere have abandoned the traditional method of isolation and, under the assumed aegis of specific reactivity, claimed that two unknowns, antibodies and proteins, interact in specific pairs simultaneously betraying each other's genesis from a virus. In other words, what is the guts of what is called HIV isolation is actually no more than a chemical reaction, an antibody test, and from an antibody test, one cannot claim proof of isolation of anything. If an antibody test is isolation of a virus then the pregnancy test, which uses an antibody to detect the placental hormone beta HCG, must be regarded as placental isolation. Of course, there may be instances of specific reactivity involving viral proteins and antibodies, but the only way to prove this is to compare reactions in the test-tube with the virus of interest. Nature would then reveal specific reactivity by the fact that reactions, the virological equivalent of curdling milk, show up only when there is virus and never if there is no virus. This is crux of the matter and where the evidence for the existence of HIV begins to fall apart. To prove specific reactivity, one must first isolate the virus for use as a gold standard for comparison. One cannot adopt specific reactivity as a premise to prove the existence of a virus if one must first isolate the virus to prove the premise upon which isolation is contingent. Try as you will but the cart does not go before the horse and the Gallo argument is reductio ad absurdum.

This leaves us in a perilous quandary. What are these unknown antibodies to unknown proteins which we call being HIV positive? They could represent a virus, but that remains to be proven by isolating a retroviral-like particle and proving it is a retrovirus. It is certainly not cogent to argue that the conjunction of a number of unspecific phenomena makes one possibility a definite outcome any more than claiming that ten men, all dressed in white, hitting a ball around a paddock, must be playing cricket. They might just as well be Ku Klux Klaners playing baseball. If not a virus, then what? If someone tests positive, is this proof that a virus has been transmitted? Or is it something altogether different? Whatever these reactions mean, they do seem to be a marker for AIDS in the high risk groups, but are they just as significant in those at low or at no risk? Does just knowing you're HIV positive affect your health? Does your doctor knowing you're positive lead to treatments for a virus you may not have? Could such treatments themselves cause harm? We now know that antibodies to the germs that cause the diseases present in 90% of AIDS patients also react with the so called HIV proteins. (Muller et al., 1991; Kashala et al., 1994) Are we being fooled by antibodies that have nothing to do with a retrovirus? Are we seeing curdle from a different milk? Why, in one study, did 10% of 1300 individuals at low risk for AIDS including blood donors have antibodies to a sufficient number of HIV proteins to deem them HIV infected by the most stringent United States criteria? (Lundberg, 1988) Why do 30% of individuals transfused with HIV negative blood develop antibodies to the same p24 protein nearly every HIV researcher uses to "isolate" HIV? (Genesca et al., 1989) Why do 50% of dogs have antibodies to one or more of these same proteins? (Strandstrom et al., 1990) How come healthy, non-HIV-infected mice injected with blood from similar mice, or mice injected with extracts of a common human bowel bacterium, develop some of the same antibodies? Why does transfusion of one's own, irradiated blood produce the same antibodies? (Kozhemiakin & Bondarenko, 1992) If these data do not mean that HIV antibodies are non-specific, then there must be some completely unknown as well as very peculiar ways for men, dogs, and mice to acquire HIV infection. On the other hand, if some humans, injected with their own or someone else's blood, or mice injected with foreign cells and foreign proteins develop "HIV antibodies" but are not infected with HIV, why should gay men, IV drug users, and haemophiliacs, who are all exposed to foreign cells and/or foreign proteins, not also develop "HIV antibodies" and not be infected with HIV? Is it possible that we been misled by non-retroviral phenomena altogether? This would not be the first time. Over the mid- to late-1970s, Gallo and his colleagues claimed to have isolated the first human retrovirus, HL23V, from patients with various types of leukaemia and their evidence included a picture from a density gradient. (Gallagher & Gallo, 1975; Gallo et al., 1976) Soon enough antibodies to the HL23V proteins were found to be widespread, even amongst normal people and there was great excitement that a cause of leukaemic was at last in the offing. However, two groups of researchers then found that the antibodies were in reality directed against a wide range of naturally occurring substances, thus destroying that particular notion of specific reactivity. (Barbacid et al., 1980; Snyder & Fleissner, 1980) Overnight, HL23V vanished from the scientific literature, so much so that Gallo now never mentions it. Does a similar fate await HIV? Neville Hodgkinson (Hodgkinson, 1996), the former science and medical correspondent for the London Sunday Times, has suggested that HIV is the greatest scientific blunder of the twentieth century. If so, there are alternative theories and therapies for AIDS we would do well to consider.

Now, are you ready for that scientist? His name is Galileo Galilei, a man no stranger to heresy. Perhaps we should heed his counsel and begin to trust our own sparks. I say the sooner the better. *

References

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