No Retroviruses Can Cause AIDS, Virus Isolation
Expert Etienne de Harven Concludes

By Paul Phillpot

Reappraising AIDS Nov./Dec. 1998

Physician Etienne de Harven was a successful cancer researcher and retroviral expert when he read in 1981 about the first four cases of what would later be called AIDS. He watched as public hysteria over the possibility of an infectious cause of AIDS transformed into a palpable demand that scientists find one.

When National Institutes of Health official Robert Gallo answered the demand in 1984 by proposing a retroviral cause, de Harven's concern turned to disappointment. He feels that the near universal acceptance by scientists and physicians of Gallo's hypothesis occurred only because they did not properly study it.

Although concerned and disappointed, this did not surprise de Harven. He had in the 1970s watched Gallo and other retrovirologists undermine the scientific standards of their profession to make it seem that retroviruses could cause a non-infectious disease, cancer, in humans. That enabled retrovirologists to become the principal beneficiaries of federal cancer research dollars.

It also set the stage for their eventual takeover of the emerging AIDS research budget, which was growing rapidly due to public fear of a contagious epidemic.

In essays published in the spring issue of Continuum and this issue of RA, de Harven makes plain his objection to the popular view of AIDS as a virus-induced condition. He states that retroviruses, including HIV, lack a capacity to cause immune deficiency, and endorses UC-Berkeley retrovirologist Peter Duesberg's assertion that such factors as narcotics and HIV drugs like AZT provide much better explanations for AIDS. He also articulates support for Australian biophysicist Eleni Papadopulos-Eleopulos' argument that HIV's existence has never been properly established, and that all the HIV tests are invalid.

Sterling credentials

De Harven's public alliance with the AIDS Reappraisal movement represents a major endorsement due to his sterling credentials. In 1953, he graduated prima cum laude with his MD from the University of Brussels, Belgium. As a research fellow, first at the Institut Gustave Roussy, in Villejuif, France, and immediately afterwards at the Sloan Kettering Institute in New York, he rapidly made two contributions to viral research. In 1958, he and Sloan-Kettering's Charlotte Friend published the first electron micrographs (pictures taken with an electron microscope) of the Friend leukemia virus (FLV), a retrovirus just discovered by Friend in murine (mouse) leukemia cells. In 1960, he demonstrated with electron microscopy that the assembly of such retroviral particles occurs on the host cell's outer membrane. In describing the steps leading to the release of retroviruses from host cells, he coined the term "budding," which has become a staple in the vocabulary of freshmen microbiology students.

In 1962, he joined the staff of the Sloan-Kettering Institute as its chief of Ultrastructural Research Section, in a joint appointment as Professor of Biology at Cornell. His laboratory became an international center for ultrastructural studies of retroviruses. In 1981, he moved to Canada, to lead the Electron Microscope Laboratory at the University of Toronto Pathology Department, while serving as a staff pathologist at Toronto General Hospital. He retired from both posts in 1993, and transferred to southern France, where he continues his affiliation with the University of Toronto as emeritus professor of pathology.

De Harven spent most of his research career characterizing and isolating murine retroviruses, using filters and ultracentrifuges to purify the particles, and electron microscopy to monitor the level of success of purification and to study viral morphology. His extensive publication record led him to serve as associate editor of Virology and Cancer Research, and as editorial board member of Scanning Microscopy, Submicroscopic Cytology, and the Journal of Electron Microscopy Technique.

Doubting HIV

De Harven doubted the infectious HIV-AIDS model from its inception in 1984, when Gallo presented it in four papers published together in the May 4 issue of Science. Among the evidence supporting his conclusion, Gallo claimed to have isolated a new retrovirus, later called HIV, from 34% of the AIDS patients that he examined. This seemed to be a very week correlation, since 66% of the patients lacked enough virus to isolate.

As for the patients in whom Gallo did claim to obtain retroviral isolates, the evidence appeared very weak as well. Although retroviruses infect immune cells, they don't kill them, so de Harven viewed them as unlikely candidates for causing AIDS. Was Gallo proposing that his new retrovirus killed host cells when replicating? No. In the cultures where Gallo claimed replication of the retrovirus take place, hosts cells survive indefinitely.

Also, Gallo attempted to isolate his retrovirus only from supernatants (cell-free culture fluid) of stimulated cell cultures, rather than from fresh patient's plasma. Why was this? Could it be that there wasn't enough virus in the plasma to isolate it in the first place? After all, culturing causes viral populations to increase beyond the levels found in vivo. "If there was enough virus to isolate directly from the plasma, as the current concept of alleged 'viral load' suggests," de Harven says, "Gallo could have done so very easily, and it would have made his fabulous story much more convincing."

Then there were the replication data Gallo used to demonstrate that these samples which he called retroviral isolates indeed contained viruses. Gallo added the primary "isolates" (the ones made from supernatants of cultures to which he had added patient tissue) to virgin cultures, which he then spiked with powerful stimulants. He showed that from the resulting secondary supernatants he could obtain a continuous supply of "retroviral isolates" identical to the originals, indicating that something in them had the capacity to replicate, a fundamental feature of viruses.

But since stimulants can induce culture cells to release all sorts of material and particles, and since both the primary and secondary "isolates" resulted only from stimulated cultures, de Harven wondered if these "isolates" were merely artifacts of culture stimulation.

There were other reasons to doubt that these samples were viral isolates. A retroviral isolate should contain a single species of RNA molecule, and it should code for the proteins in the sample.

Although Gallo showed that his "isolates" contained RNA molecules and several proteins, including the enzyme reverse transcriptase, he didn't show that all the RNA molecules were identical, or that any one of them coded for the proteins. Instead, he concluded that the molecules were viral simply because some of the proteins reacted to antibodies in the fresh (uncultured) plasma of 88% of his AIDS patients, and because he assumed incorrectly, de Harven says that reverse transcriptase is an exclusive feature of retroviruses.

Gallo's electron micrography data particularly intrigued de Harven, as this involved his specialty. He says that a convincing and proper demonstration of the existence of a retrovirus should contain two sorts of electron micrographs. One should show retrovirus-looking objects of identical size and appearance in uncultured patient tissue samples; the other should show isolates of those objects, meaning samples that exclusively contain objects having identical size and appearance to each other and the objects observed in the tissue. Instead, Gallo presented micrographs of stimulated cultures to which he had added AIDS patient immune cells or plasma, but no micrographs of his "isolates." The micrographs of the cultures showed cells along with spherical objects nearby of various sizes which Gallo claimed where representatives of his new retroviral species.

To this day, de Harven says he can find no studies that have improved upon Gallo's electron microscopy evidence. No one has claimed to have produced electron micrographs of retrovirus-looking objects in the uncultured plasma of a single AIDS patient, or to have obtained "HIV isolates" from uncultured plasma. De Harven calls this "a totally unacceptable contradiction with the alleged PCR measurements of 'viral load' in these patients."

Only in 1997 did researchers two teams, in fact finally publish electron micrographs of so-called "HIV isolates" (Virology 230:125 and 134) made from stimulated cultures derived from AIDS patients' blood. As de Harven suspected, the published pictures showed mostly non-viral material, i.e. a heterogeneous mixture of debris plus, he says, "particles that resembled cellular fragments but had no resemblance to retroviral particles."

The authors labeled some of these particles HIV and stressed that control cultures cultures unexposed to AIDS patients' blood produced no such particles. "But those 'control' cultures had not been stimulated and, therefore, are questionable," de Harven says.

The Good Old Days

De Harven says when he began his career in 1953, claims of viral isolation and causation were much more convincing, and had been so since the birth of virology, in 1892.

That year a Russian bacteriologist discovered he could induce mosaic disease in healthy tobacco plants by injecting them with fluid from sick plants. Since the fluid didn't contain any of the diseased tissue, he concluded that there was some exogenous causative agent. He showed that the agent was microbial rather than molecular, since microbes can sustain their populations within an organism via replication, whereas exogenous molecules can do so only via regular replenishment from an outside source. He also established that this microbe was invisible with the light microscope and smaller than any known bacteria or fungi the only known microbes at that time by reproducing the results after passing the fluid through special filters with ultramicroscopic pore sizes (a process called ultrafiltration).

In 1898, a Dutch scientist found that this filterable infectious agent was an unknown type of microbe, since it remained active even when subjected to challenges that would destroy bacteria and fungi. He called it a virus. Using similar techniques based on ultrafiltration, scientists found viral causes for other diseases, including many animal and even human diseases. In 1956, Friend demonstrated the existence of a mouse leukemia (immune cell cancer) virus by inducing the disease in adult Swiss mice with injections of ultrafiltered tissue extracts from leukemic laboratory mice.

Scientists learned to approximate the size of viruses by using filters of different and relatively well-calibrated pore diameters. This enabled Friend to demonstrate that her virus, like all other viruses then shown to cause cancer in lab animals, probably had a diameter close to 100 nanometers (nm).

Such techniques also enabled scientists to purify viral samples according to size, to remove entities that are much larger or smaller than their virus, in attempts to produce viral isolates. De Harven and Friend followed this standard procedure to obtain purified samples of their retrovirus.

The next step of the standard procedure called for them to produce micrographs of their purified samples, to see how close they had come to ideal isolation, and to document the size and morphology of the objects in their sample. Their micrographs showed very little else but spherical objects of the same size (100 nm) and morphology. The objects looked exactly like those in electron micrographs of the leukemic mouse tissue from which they'd abstracted their filtrates. The objects also closely resembled the viruses shown to induce cancer in other laboratory animals.

In the 1970s, purification by ultrafiltration gave way to purification by density. This process involves placing a sample on a sucrose gel that has a graduated density, meaning that from top-to-bottom the gel's density increases from light to heavy. By spinning the gel in a high-speed centrifuge a process called ultrafugation the sample's contents settle at their characteristic densities, forming bands that constitute density-purified samples.

De Harven and Friend showed that Friend's virus, like all those associated with cancers in lab animals, banded at 1.16 grams per millimeter (gm/ml).

"Collecting the 1.16 band is a very efficient method of concentrating retroviruses," de Harven explains. "Retroviruses concentrated by this method look similar, under the electron microscope, to those concentrated by the old ultrafiltration method. The degree of purity of these retroviral concentrates depends on the amount of cellular microvesicles and debris contaminating the initial sample which may very well sediment at the same density of 1.16 gm/ml."

Other methods came along to help scientists to study viruses.

Growing cells in cultures and infecting them with viral isolates permitted virologists to distinguish two general groups of viruses. When some viruses, like those associated with polio, influenza, and herpes, replicate inside culture cells, the cells quickly die. Other viruses, including those associated with cancer, lack this cytolytic capacity, and replicate without killing their hosts. The viruses associated with cancerous lab animals, including Friend's, actually make the culture cells become cancerous, a process called transformation or immortalization, adding support to the contention that they cause cancer in vivo.

Virologists also developed a way to study the proteins and gene molecules RNA or DNA of viruses. By using chemicals that break viruses apart, and a process called electrophoresis that separates molecules according to molecular weight, scientists can turn a 1.16 gm/ml retroviral isolate band into several new bands, each one representing an isolate of a different molecular species that composes the virus.

Logically, this process applied to a retroviral isolate should produce a single band with RNA molecules in it, and those molecules should be identical to each other and code for the proteins that form the other bands.

Even before the advent of this technology, Friend showed that her virus, like the others associated with cancer, contained RNA, which is why these viruses were called "RNA Tumor Viruses."

In 1970, scientists found that the protein forming one of the electrophoresis bands from retroviral isolates was an enzyme capable of reverse-transcribing RNA molecules into DNA molecules. They named this enzyme reverse transcriptase, and gave RNA tumor viruses the new name retroviruses.

That same year Duesberg showed that these viruses caused cancer because their RNA molecules contained a special extra onc-gene. This contrasted with the cyctolytic viruses, which other scientists found contained a gene that coded for a cytolytic enzyme responsible for destroying the host cell's membrane.

Isolation standards

De Harven says that the collective lessons of retrovirology by 1970 led to an important fundamental conclusions: a 1.16 gm/ml band qualified as a retroviral isolate only after electron microscopy showed that it looked like one, and unstimulated cultures showed that it behaved like one. This is because on many occasions virologists obtained 1.16 gm/ml bands that flunked the electron microscopy test, including bands that contained nothing at all that even vaguely resembled viruses. And some 1.16 gm/ml bands that did resemble retroviral isolates failed to demonstrate any replicative capacity at all, and therefore qualified as isolates of non-viral objects that resembled retroviruses.

In other words, everything that looks like a retrovirus is not necessarily a retrovirus, and everything that looks like a retroviral isolate is not necessarily a retroviral isolate.

De Harven says this conclusion holds even for 1.16 gm/ml bands that contain reverse transcriptase, which virologists have "found not to be unique for retroviruses, but instead a normal constituent of many cells."

No retroviral isolates from humans

The retrovirus-cancer link demonstrated among special inbred lab animals caused scientists to wonder if retroviruses might explain some forms of human cancer.

This idea initially interested de Harven. But he and some other retrovirologists began to doubt that retroviruses could cause human cancer.

For one thing, the retrovirus-cancer link in animals was reserved for special cases of inbred mice, chickens, and cats, and even then the link was far from perfect. Among these peculiar laboratory animals, scientists could usually isolate retroviruses from the subjects that had cancer, but sometimes they could not. And sometimes they could isolate retroviruses even in the absence of cancer. Furthermore, these retroviral isolates would not induce cancer when injected into wild mice and chickens. Nor could anybody isolate retroviruses from any wild animals, or even other laboratory animals, besides special inbred cats.

Nonetheless, the entire profession in 1955 set its sights on isolating retroviruses from humans suffering from various types of cancers. By 1970, the examination of many cancerous human tissues resulted in some electron micrographs that showed mixed in with the cells and other material a few particles "with a vague resemblance to retroviruses," but no micrographs of isolates. "Unfortunately," de Harven says, "the term 'virus-like particles' kept being described in the literature, wrongly perpetuating the notion that actual retroviruses were occasionally associated with some human cancers."

Typical retroviruses objects of identical size and appearance, isolates of which contain a single RNA species that codes for the proteins in the sample were never conclusively demonstrated for humans. "This was in sharp contrast to the highly reproducible demonstration, by electron microscopy, of typical retroviruses in a variety of mouse and chicken leukemias and tumors."

Gallo lowers the standards

Nonetheless, prior to HIV's official birth in 1984, Gallo published the only three claims for isolation from human subjects of retroviruses, each one of which he said caused a different form of human leukemia. His claims flunked the strict criteria established previously by scientists demonstrating the existence of retroviruses in lab animals, and causally linking those retroviruses to cancer.

Gallo's "evidence" resembled what he'd later use to demonstrate HIV's existence and causal role in AIDS: the presumption that all 1.16 gm/ml bands that contain reverse transcriptase are "retroviral isolates"; the presumption that anything that vaguely resembles a retrovirus is a retrovirus; the use of stimulated cultures; micrographs of those cultures, but not of the density-purified bands; the failure to obtain them from uncultured plasma; the failure to use unstimulated cultures; the failure to obtain these bands from most of the patients examined; the failure to demonstrate that these bands affect virgin cultures in a way that explains the disease (rather than transforming the culture cells, Gallo's 1.16 gm/ml bands required that the culture cells already be cancerous); the failure to demonstrate that his bands contained a single RNA species and that it coded for the proteins in the band; the failure to demonstrate that the RNA contained a gene in this case, an onc-gene that could explain the disease.

"Why no micrographs of the 1.16 gm/ml bands themselves?" de Harven asks. "Most probably, Gallo's electron microscopy results from his 'isolates' were negative and swiftly ignored."

Yet a large segment of the biomedical profession, eager to consolidate the hypothetical role of human retroviruses in human diseases, accepted Gallo's new standards. Though one of his first claims was eventually discarded, the pump was primed for the retroviral hunt among AIDS patients.

"The faith in retroviruses as pathogens assumed quasi-religious proportions," de Harven laments. "Since electron microscopy could not demonstrate viruses in the 1.16 bands from human subjects, we forgot about microscopy and started relying on 'markers.' Microscopy is time-consuming and skill-demanding. Who has time for that? Not when research funding was getting difficult and when major pharmaceutical corporations were starting to finance 'crash programs' for speedy answers.

"When retroviruses are legion, molecular markers provide a useful approach to quantification probably better than direct particle counting under the electron microscope (which I always found difficult). But without isolates, the use of markers is methodological nonsense. 'Markers' of what? We know that all of the so-called 'HIV markers' are totally non-specific." *