Retrovirologists, retroviruses and purification 1 July 2004
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Eleni Papadopulos-Eleopulos,
Biophysicist
Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala

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Re: Retrovirologists, retroviruses and purification

Retrovirologists, retroviruses and purification

 

In his rapid response "Re: Re: More on the photo", 25th June Brian Foley wrote: "To make claims that there are certain rules for virus isolation, when in fact those rules do not and never did exist, is lying.  If the parties listening to those lies believe them, the health of a great many people is at risk".

 

As in any other branch of science, in retrovirology there are scientific principles and methods and rules under which retrovirology is practised.  According to one of them, which is nothing more than commonsense, to claim that a protein is a retroviral protein or a fragment of RNA is the genome of a retrovirus, evidence must exist that the protein or RNA originated from viral particles.  If there is no proof that the "HIV" proteins originated from retroviral particles it would not be possible to have "HIV" serology.  If there is no proof that the "HIV" RNA originated from retroviral particles it would not be possible to claim the existence of the "HIV" genome, probes, clones and primers.  That is, there would be no proof for the existence of HIV.

Until the advent of density gradient ultracentrifugation, the electron microscope and certain advances in immunology and lately molecular biology, retroviruses were defined by filtration and their biological properties.  The latter were the induction of tumours by the filtrates hence their name oncoviruses or “filtrable agents”.

 

As far back as 1957 one well known retrovirologist, J W Beard, pointed out that the use of filtration as a method to purify retroviruses was less than satisfactory.  Neither was the finding of particles using the then new method of electron microscopy sufficient to prove the existence of a retrovirus.  Beard stressed: "identification, characterisation, and analysis are subject to well-known disciplines established by intensive investigations, and the possibilities have by no means been exhausted.  Strangely enough, it is in this field that the most frequent shortcomings are seen.  These are related at times to evasion of disciplines or to their application to unsuitable materials.  As was foreseen, much of the interest in the more tedious aspects of particle isolation and analysis has been diverted by the simpler and undoubtedly informative processes of electron microscopy.  While much can be learned quickly with the instrument, it is nevertheless clear that the results obtained with it can never replace, and all too often may obscure, the need for the critical fundamental analyses that are dependent on access to homogenous materials"1(emphasis ours).

 

In 1972 Toplin wrote:  "As part of the Special Virus-Cancer Program of the National Cancer Institute, we have been applying the Model K and other zonal centrifuges to the concentration and purification of a variety of murine, feline, and avian ribonucleic acid (RNA) tumor viruses propagated in cell culture.  This report is a summary of the techniques used in our laboratory to prepare highly purified concentrates of these relatively high-yield viruses.  The same techniques have been used for the recovery of other viruses, particularly the low-yield viruses found in cell cultures of human origin such as the ESP-1 type C virus and the Epstein –Barr  herpesvirus".  One year later, Barre-Sinoussi and Chermann pointed out the presence in their purified preparations of particles with "No apparent differences in physical appearance"2(emphasis ours).

 

In 1985, Donald Francis, one of the originators of the retroviral theory of AIDS wrote:  "One must rely on more elaborate detection methods through which, by some specific tool, one can 'see' a virus.  Some specific substances, such as antibody or nucleic acids, will identify viruses even if the cells remain alive.  The problem here is that such methods can be developed only if we know what we are looking for.  That is, if we are looking for a known virus we can vaccinate a guinea pig, for example, with pure virus…Obviously, though, if we don't know what virus we are searching for and we are thus unable to raise antibodies in guinea pigs, it is difficult to use these methods…;  we would be looking for something that might or might not be there using techniques that might or might not work"3 (emphasis ours).

 

In 1975 John Bader wrote:  "A variety of experiments addressed to the elucidation of the structure of RTV [retroviruses], or particularly in the use of RTV or components of RTV as reagents, require some confidence in the relative purity of the RTV preparation…RTV have a characteristic buoyant density, and centrifugation to equilibrium in density gradients is the preferred technique for purification of RTV" 4(emphasis ours).

 

In 1997 Pablo Gluschankof, Hans Gelderblom and their colleagues wrote:  "Virus ["HIV"] to be used for biochemical and serological analyses or as an immunogen is frequently prepared by centrifugation through sucrose gradients.  The fractions containing viral antigen and/or infectivity are considered to contain a population of relatively pure virus particles"5 (emphasis ours).

 

In the same year, 1977, Montagnier stated:  "….analysis of the proteins [and thus of the genome] of the virus demands mass production and purification.  It is necessary to do that"6 (emphasis ours).  Thus retrovirologists and "HIV" experts (Brian Foley and Christopher Noble are exceptions) agree that for characterisation of a retrovirus (of its protein and genome) purification is necessary.

 

The question is how pure should pure be.

 

In an email exchange between Robin Weiss and us he wrote:  "I do not think Barre-Sinoussi et al misled the scientific community by calling the 1.16 g/ml band purified virus.  But if you and your colleagues prefer to call it enriched but not yet completely pure, I would happily concur with that opinion.  This illustrates what I mean by a sterile argument:  how pure is pure?  Is distilled water 'pure'?  Yes, but it will still have a few parts per million or per billion of other soluble molecules". 

 

Our response was: "We cannot call Barre-Sinoussi's 1.16 g/ml band pure or even "enriched" since the scientists who prepared the material conceded it contained no retrovirus-like particles, much less the particles of a unique retrovirus…Consider your analogy of pure water.  Suppose you are doing a vital experiment which requires pure water.  Your colleagues on the floor above provide a bottle labelled "pure water" which forms a critical part of your experiment and ultimately the results of this experiment are used in a way which affects the lives of millions of people.  Fifteen years later you are informed that the bottle your colleagues gave you, far from being pure water, contained not even one molecule of water.  Would you not think that your colleagues were wrong to label it "pure water" and that they had misled you?  Would you then be happy to call it water enriched?…In their cultures Koch and Petri had gelatin plus particles which could be unambiguously identified as bacteria using the light microscope.  Any proteins (other than gelatin) RNA or DNA found in the culture could only be of bacterial origin…Unlike the bacteria, [Petri] dishes which in addition to bacteria contain only one known protein, gelatin, the "HIV" dishes contain cells, cellular fragments and perhaps bacterial fragments as well as mycoplasmas and viruses", which like retroviruses contain proteins and RNA.7

 

In other words, it is not important how many and what concentration of impurities are present in the "purified" viral preparations.  The important thing is that the impurities should not contain confounding molecules (proteins and RNA) and if there are such molecules present their concentration should be insignificantly small.

 

We were always aware that it is difficult to obtain a "purified" retroviral preparation which contained nothing else but retrovirus particles.  That is why from the beginning we asked to see EMs of Montagnier and Gallo's "purified" HIV.  Our expectations were to see EMs of the "purified" "HIV" similar to those of Crawford, Sinoussi and Toplin.  We would never have imagined that a preparation which contained no retrovirus-like particles or which was "purified vesicles" (as Gluschankof et al call their preparation) would be claimed and accepted as being "purified" "HIV".

 

To claim that some of the proteins in the "purified" preparations were HIV proteins and some of the RNA, "HIV" RNA ("HIV genome"), defies not only scientific reasoning but commonsense.

 

References

 

 

1. Beard JW. Physical methods for the analysis of cells. Ann N Y Acad Sci 1957;69:530-544.

2. Sinoussi F, Mendiola L, Chermann JC. 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. Francis DP. The search for the cause. In: Cahill KM, editor. The AIDS epidemic. 1st ed. Melbourne: Hutchinson Publishing Group, 1983:137-150.

4. Bader JP. Reproduction of RNA Tumor Viruses. In: Fraenkel-Conrat H, Wagne RR, editors. Comprehensive Virology. New York: Plenum Press, 1975:253-331.

5. Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ. Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virol 1997;230:125-133.

6. Tahi D. Did Luc Montagnier discover HIV?  Text of video interview with Professor Luc Montagnier at the Pasteur Institute July 18th 1997. Continuum 1998;5:30-34. www.virusmyth.net/aids/data/dtinterviewlm.htm

7. Turner V, Weiss R. Email debate with Professor Robin Weiss on the existence of HIV. 1999.  www.theperthgroup.com/EMAILCORR/vftweiss.html

 

 

 

 

 

Competing interests: None declared