'HIV' HAS NEVER BEEN ISOLATED

Guest Post translated from French

Original found here: http://www.sidasante.com/edh/edhvih.htm

Namur — October 12, 2002.

Etienne de Harven

In AIDS dissent circles, you will often hear the assertion that HIV has never been isolated. This assertion is categorically rejected by well-meaning orthodoxy as belonging to the realm of heresy. It is often debated in a somewhat laborious way by dissenters, the difficulty arising from the fact that the word "isolation" is not always given the same definition.

To better understand this debate, we are going to spend a few moments reviewing together what is meant, in classical virology, by the words “isolation” and “purification”. On the basis of which, we will conclude on the application of these terms in the specific case of HIV.

In experimental pathology, working on well-selected chickens or mice, several diseases including various forms of cancer and leukemia can be transmitted by the injection of "acellular filtrates" into these animals. Since these filtrates are totally devoid of cells and bacteria, such experiments made it possible to make a clear distinction between the transmission of a cancer by cell graft, or by infra-microscopic factors such as viruses. Such filtrates were obtained by various ultrafiltration techniques, techniques which completely eliminated the presence of whole cells or bacteria. If, in addition, the activity of such filtrates were concentrated by high-speed centrifugation, maintained after storage at low temperature, but lost by heating to 65-68°, it could reasonably be concluded that the disease in question had been transmitted by a virus. And it could be said that the incriminated virus had been "isolated". It was such a methodological approach that enabled Peyton Rous to "isolate" the sarcoma virus from chickens, and John Bittner to "isolate" the virus from mouse mammary tumors, all well before the invention and the application of electron microscopy to experimental virology.

When similar experiments are carried out, no longer on laboratory animals but on cell cultures, the "isolation" of a virus can be demonstrated, a result which is then based on the observation under the microscope of various cellular alterations, such as formation of multinucleated giant cells. And it was experiments of this type that enabled Luc Montagnier's research group at the Institut Pasteur in 1983 to isolate a retrovirus initially called LAV and renamed "HIV" shortly afterwards.

The difficulty of interpretation, in the case of the discovery of the Montagnier group, stemmed from the fact that the cell cultures used were very complex, comprising in fact a mixture of several cell types, some of which are well known as being chronic carriers of retrovirus. There was indeed "isolation" of a retrovirus, fine. But there was no evidence that this "isolation" had any connection with the infection of cell cultures with extracts from an AIDS patient. In short, there had probably been "isolation" of a retrovirus, but there was no reason to claim that this virus came from the patient, and therefore no reason to call it HIV, which brings us back to the title of my presentation: HIV has never been isolated!

But there is another problem related to the interpretation of this type of viral isolation. The problem being that this type of isolation does not in any way make it possible to identify with certainty molecules that could be considered as specific molecular "markers" of the virus.

Because, in fact, to identify with certainty molecules that could be considered as specific "markers" of a virus, this virus must first be highly purified, that is to say separated from any contamination by cellular or bacterial debris. The success of such purification must be rigorously tested, failing which the identification of so-called "markers" is a serious scientific fraud. It is here that electron microscopy takes on an essential role, because to test the success of a purification technique, and despite all the noise made around molecular biology techniques, it remains the only method which makes it possible to demonstrate that virus particles have been successfully separated from any contamination of cellular, bacterial or mycoplasma origin.

Two methods have been used successfully to purify viruses.

One is based on ultrafiltration, the other on high-speed centrifugation in density gradients.

In my research on mouse leukemia viruses (Friend leukemia), I used a combination of ultrafiltration and centrifugation methods which enabled us to demonstrate, in 1965, a remarkable degree of purification of the Friend virus ( 1). I have never used gradient techniques, although they have been used with great success by other authors whose work had clearly demonstrated that carcinogenic RNA viruses (as they were called before 1970) all sedimented, in sucrose, at the density of 1.16 gm/ml.

The problem with the gradient method, however, was clear: it was well recognized that many cellular debris, such as microvesicles, also sediment at this magic density of 1.16gm/ml. Collecting material at this density is therefore not enough to proclaim the isolation of a retrovirus, far from it! The need to check the absence of cellular debris by electron microscopy is therefore an absolute necessity, a fact which had been clearly reaffirmed in 1973, at the Institut Pasteur, during an important conference which dealt exclusively with methods of purification of retroviruses. (2).

Before considering the implication of these remarks for the alleged "isolation" of HIV, we must return to a momentous event that took place in 1970, namely the discovery by Temin (3) and by Baltimore ( 4) reverse transcriptase (ie DNA synthesis from an RNA template). It was a revolution in molecular biology to understand the activity of this enzyme which has been aptly called reverse transcriptase (RT).

In 1970, transcription from RNA to DNA was certainly a surprising discovery. A discovery that provided a very attractive explanation for the possible mechanism of action of carcinogenic RNA viruses! Reverse transcription had never been observed in biology before 1970, and the interest in this discovery was such that it was decided, shortly after, to re-baptize carcinogenic RNA viruses under the name of retroviruses...

But where was the problem?

The problem was that neither Temin nor Baltimore checked the purity of the virus samples in which they identified the enzyme activity in question.

However, shortly after their 1970 publications, it became clear that reverse transcription was quite a common phenomenon in biology, as summarized by Varmus in 1987 (5). As early as 1971 (6), it appeared that reverse transcription was common to a large number of animal cells, as well as bacteria (7).

Consequently, before considering the enzyme as a retroviral marker, it would have been necessary to repeat the experiments of Temin and Baltimore on samples whose degree of purification would have been verified, in order to exclude the presence of cellular debris which could , by themselves, explain the presence of reverse transcriptase activity. To my knowledge, these checks have never been carried out, and the enzyme has been considered for 30 years as the main marker of retroviruses!

In the historic article published by Barré-Sinoussi, Chermann, Montagnier and collaborators (8) and in which the isolation of a retrovirus was announced, the detection of the enzymatic activity (RT) in a fraction sedimenting at 1.16gm/ ml was the key to demonstrating a retrovirus. However, we now know that this enzyme is not a specific marker for retroviruses! And we have known for a long time that the 1.16gm/ml fractions contain an abundance of cellular debris perfectly capable of explaining the presence of enzymatic activity...

Barré-Sinoussi's article also made much of an electron microscopy image illustrating retroviruses budding on the surface of a lymphocyte. The image was interpreted as proof of infection of the cultured cells by the extract from the patient. What the article failed to consider was that the cultures were mixed with lymphocytes from umbilical cord blood, and that the human placenta had been known for several years (9) to be a tissue exceptionally rich in endogenous retroviruses ( HERVs).

In short, the article considered worldwide as the basic reference on HIV isolation is based on three methodological errors:

1) Not having verified the presence of cellular debris in the fractions,

2) having ignored the enzymatic activity of these same cellular debris, and

3) having ignored the presence of endogenous retroviruses in the cells in culture. This article can be considered as the demonstration of a retrovirus, probably endogenous to the cell cultures used. But it cannot be presented as proof of the isolation of a retrovirus from an AIDS patient.

It took 15 years for the first experimental controls to be carried out, in two laboratories, one in the United States (10), the other in France (11).

These two laboratories jointly published, in Virology, their results of study under the electron microscope of gradients obtained from cell cultures supposed to produce HIV. In both cases, the authors observed an abundance of cellular debris, with no acceptable evidence of retroviral particles.

Around the same time, Luc Montagnier was interviewed by Djamel Tahi and ended up admitting that, indeed, HIV had never been purified in his laboratory… (12).

It is interesting to note that in the article originating from Pasteur in 1973, it was clearly indicated that reverse transcriptase activity is present in cellular debris. As incredible as it may seem, it was in this same laboratory of the Institut Pasteur that, ten years later, in 1983, the role of cellular debris was ignored, putting AIDS research on the wrong track for the 20 years to come...

Before concluding, I would like to add a few remarks relating to other molecular "markers", also relating to the alleged genomic isolations based on PCR techniques, and finally relating to the abuse of the credibility of the public by images, embellished by computers, which are said to represent HIV.

Other molecular markers

Let's now go back to reverse transcriptase, which has already largely caught our attention.

Several proteins, allegedly of retroviral origin, are frequently used as specific "markers", for example p24. The doubt, already old, on the specificity of this marker was clearly analyzed recently by Fabio Franchi (13) who underlines the absence of any correspondence between the results obtained with p24, and the measurements of the so-called "viral load" supposed to be measured by PCR. On the other hand, one cannot fail to be troubled to learn that 50% of dogs, tested by Western blot, react positively with one or more of the HIV proteins obtained by genetic recombination, such as gp120, gp41, p31, and p24 (14).

The lack of specificity of these so-called HIV structural proteins was clearly demonstrated nearly 10 years ago by Eleni Papadopulos and the Perth group in a classic paper published in 1993 in Nature/Bio-technology (15). The conclusions of these authors were crystal clear, but have been carefully ignored by AIDS orthodoxy. A constituent of normal cells such as actin probably corresponds to gp41, while gp120-160 probably corresponds to an oligomer of gp41.

Obviously, the presence of cellular debris easily explains the presence of so-called retroviral "markers". And all the so-called successes in HIV isolation are easily explained by abusive applications of non-specific markers. As we have already pointed out, Gene markers and viral load measurement by PCR.

This approach could, in principle, seem more attractive for two reasons:

1) it applies directly to the blood of patients, thus avoiding all the uncertainties that hover around cell cultures, and

2) the methods are supposedly quantitative.

And however…

Note that it has never been possible to visualize, under an electron microscope, the slightest retroviral particle in the blood of AIDS patients, even if patients with a very high viral load are selected (16). In addition, it seems probable that PCR techniques are capable of amplifying small RNA fragments originating from gene fragments of endogenous retroviruses, which would be expressed more abundantly under stress conditions. You should know that more than 2% of the human genome is represented by endogenous retroviral sequences (17, 18). Measuring so-called "viral load" by PCR may well have nothing to do with the quantitative measurement of so-called exogenous HIV. At last, let's not forget the absence of any correlation between so-called viral load measurements and those of the number of molecules of p24 in the circulating blood of patients. Let's not forget either that Karry Mullis himself, the discoverer of the PCR technique who received the Nobel Prize for it in 1993, categorically rejects any application of "his" PCR technique to quantitative measurements of HIV...(19 ).

The abuse of beautiful images

You can find, in newspapers and magazines around the world wonderful images, high in totally artificial colors, which are supposed to represent HIV itself, embellished by computers. To publish such images is to bring to the attention of the general public and doctors an apparently limpid and clear message: HIV has indeed been isolated since it can be seen and portrayed under an electron microscope!

This is a huge lie!

All these images come from cell cultures. None comes directly from a single AIDS patient (20), even if we try to select patients labeled as having a high viral load.

Luc Montagnier himself described the very complex cell cultures used for HIV as real soups of retroviruses (12)! And it is very true! This is true, because everything had been planned for retroviral particles to appear there, and because the elementary checks which should have made it possible to understand the real origin of these viruses were never done, or if they were done have never been published!

These cell cultures are always mixed and hyper-stimulated.

Mixed, because they consist of a clever mix of several cell lines including, for example, the patient's lymphocytes plus Gallo's H9 cells, cells which are well known as chronic carriers of retroviruses (21), or the patient's lymphocytes mixed with lymphocytes from the umbilical cord which, derived from the placenta, have every chance of carrying endogenous retroviruses. An example of misuse of the convincing force of a beautiful image is provided by the classic article by Barré-Sinoussi, Pasteur 1983. It shows an excellent image taken with an electron microscope and representing particles of retrovirus budding on the surface of a lymphocyte. Perfect ! But the authors use this image to prove that this lymphocyte was infected by the patient's viruses.

However, nothing proves this interpretation! Everything suggests, on the contrary, that the endogenous retroviruses of this lymphocyte from the blood of the umbilical cord were activated by the particular conditions of the culture.

All these mixed cultures are, moreover, hyper-stimulated by different growth factors such as phytohemagglutinin (PHA), T lymphocyte growth factor (TCGF), plus Interleukin-2, or even more corticosteroids. However, all these factors are known as activators of the expression of the endogenous retroviruses that we all carry within us (18). Should we therefore be surprised to observe retroviral particles in such hyperstimulated "retroviral soups"? No, definitely not.

It is very disturbing to note that in this historical article from 1983, electron microscopy was not used when it was essential to identify cellular debris, and was misinterpreted in the case of umbilical cord lymphocytes…

Conclusion

Indeed, HIV has never been isolated or purified.

Retroviral particles, most likely of endogenous origin, have been observed in cell cultures, but their hypothetical link with AIDS patients has never been proven, hardly more than their pathogenicity.

For political and non-scientific reasons, reasons on which we have explained ourselves this morning, AIDS orthodoxy has tried to face up to this difficulty of directly isolating the virus by inventing several molecular "markers". Because the HIV=AIDS hypothesis had to be saved at all costs (22), even at the cost of scientific integrity (23)! We have seen, however, that these markers, which totally lack specificity, did not lead to any coherent observation.

If AIDS was indeed a disease caused by a retrovirus, how is it that 20 years of research have failed to isolate the exogenous retrovirus responsible? How is it that what was so easily demonstrated in mice is so difficult to demonstrate in humans?

Twenty years of effort based on a single hypothesis, the HIV=AIDS hypothesis. Twenty years of effort to achieve, in 2002, no curative treatment, no vaccine, and no verifiable epidemiological prediction...

Don't you think it is high time to courageously ask the essential question? The question being: is the HIV=AIDS hypothesis correct? Because there is a way to see AIDS differently, outside the restricted framework of infectious diseases and retrovirology. Think of toxicology, pharmacology, malnutrition, stress...

And in this perspective, which is loaded with optimism, the difficulties encountered in the efforts to isolate and purify the so-called HIV find a simple and totally disarming explanation: these difficulties probably result from the fact that HIV DOES NOT EXIST as an exogenous and infectious agent. The doubt about the very existence of HIV has been raised by several scientists for several years (24, 25). But, for orthodoxy, it was necessary to remain politically correct, even if it was necessary to invent HIV to try to justify large investments, to develop enormous pharmaceutical markets, and also… to save face!

Don't forget the title of the book that the father of AIDS dissent, Peter Duersberg, published in 1996. Its title was: "Comment on a INVENTÉ le virus du sida", in English "Inventing the AIDS Virus"...

References

1. de Harven E. Viremia in Friend murine leukemia: the electron microscope approach of the problem. Pathology-Biology 1965; 13:125-134.

2. Sinoussi F et al. Purification and partial differentiation of the particles of murine sarcoma virus (M. MSV) according to their sedimentation rates in sucrose density gradients. Spectra 1973; 4:237-243.

3. Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 1970; 226:1211-1213.

4. Baltimore D. RNA-dependent DNA polymerase. Nature 1970; 226: 1209-1211.

5. Varmus H. Reverse transcription. Science. Am 1987; 257:48-54.

6. Ross J et al. Separation of murine cellular and murine leukemia virus DNA polymerase. Nature New Biology 1971; 231:163-167.

7. Beljanski M. In vitro synthesis of DNA on an RNA template by an Esscherichia coli transcriptase. CR Acad. Science. 1972; 274: 2801-2804.

8. Barré-Siunoussi F. et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983; 220: 868-871.

9. Panem S. C type virus expression in the placenta. Curr Top Pathol 1979; 66:175-189.

10. BessJW et al. Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 1997; 230:134-144.

11. Gluschankof P et al. Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 1997; 230:125-133.

12. Djamel Tahi. Did Montagnier discover HIV? "I repeat, we did not purify!". Continuum 1997; 5:30-34.

13. Frenchi F. In search of HIV. http://www.cesil.com/0898/bifrah08.htm

14. Strandstrom HV et al. Studies with canine sera that contain antibodies which recognize human immunodeficiency virus structural proteins. Cancer Research 1990; 50:5628s-5630s.

15. Papadopulos-Eleopulos E et al. Is a positive Western blot proof of HIV infection? Bio/Technology 1993; 11;696-707.

16. de Harven E. Summary statement. Interim Report of the AIDS avisory panel, Pretoria, May 2000. Published by the South Africa government on April 4, 2001.

17. Urnovitz HB et al. RNAs in the sera of Persian Gulf War veterans have segments homologous to chromosome 22q11.2. Clin Diagn Lab Immunol 1999; 6/3: 330-335. See also http://www.chronicillnet.org

18. Löwer R et al. The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc Natl Acad Sci USA 1996; 93:5177-5184.

19. Mullis K. "Dancing Naked in the Mine Field". Pantheon, 1998

20. Gelderblom HR. HIV sequence data base: fine structure of HIV and SIV. http://hiv-web.lanl.gov/HTML/reviews/Gelderblom.html

21. Dourmashkin RR et al. The presence of budding virus-like particles in human lymphoid cells used for HIV cultivation. VIIth International Conference on AIDS. Florence 1997:122.

22. Weiss R, Wain-Hobson S. The Durban Declaration. Nature 2000; 406:15-16.

23. Stewart GT et al. Not all accept the Durban Declaration. Nature 2000; 407; 286.

24. Papadopulos-Eleopulos E. A brief history of retroviruses. Continuum 1997; 5:25-29.

25. Lanka S. HIV; reality or artefact? Continuum 3, #1,4-9, April-May 1995.

26. Duesberg P. "Inventing the AIDS virus". Regnery Publishing, Inc., Washington, DC 1996.

Original post here: http://www.sidasante.com/edh/edhvih.htm