A blood sample suspected of containing a pathogenically significant quantity of cell-free retroviruses is centrifuged to spin out the red and white blood cells, leaving just the plasma, the basic fluid in which the cells float. The fresh plasma is then diluted 1/1 with cold heparinised Ringer's solution. Heparin is an anticoagulant, added to thin the plasma and render it more suitable to pass successively through two millipore filters of diminishing size, first using 0.6 then 0.22 micrometer pore size diameter filters. These filter out any debris larger than the size of the viral particles being sought. The last filter used will be slightly larger than the 100-120nm diameter of a retrovirus. The final filtrate is then spun in a centrifuge at 30,000 g for two hours. This has the effect of concentrating any virus in the plasma into a tiny pellet which can be recovered from the bottom of the test tube. The pellet is then fixed, embedded in an epoxy resin block and shaved into ultra thin slices which can be mounted on copper grids and examined under EM.

Figure 1. Electronmicrograph of densely packed parti cles with retroviral morphology identified as the Friend murine (mouse) leukaemia virus. Pathologie-Biologie, Vol. 13, pp. 125-134

A perfect illustration of de Harven's purification technique is seen in Fig.1. This electron micrograph by de Harven was published in 1965, (Pathologie-Biologie, Vol.13, pp. 125-134) and shows densely packed particles with retroviral morphology identified as the Friend murine (mouse) leukaemia virus. The densely packed particles, identical in shape and size, were pelleted down from fresh plasma using the method described. The magnification is 19,500 x and it can be seen that there is very little extraneous material contaminating the final pelleted isolate, indicated by three arrows. The Friend virus is classified morphologically as a Type C oncovirus, one of a small group of retroviruses which encode a cancer-causing gene in their RNA. The ultra thin slice through the pellet clearly shows the dense core in bisected virions, as round black dots.

As Dr. de Harven explains in his second Continuum article, even by the late 'sixties there was a growing tendency to abandon the use of EM, on the pretext that it was cumbersome and time-consuming, in favour of the purification and identification of possible viruses using other means, chiefly the use of the sucrose density gradient without EM, allied with indirect surrogate markers. Already, the virologists were starting to cut corners. Moreover, after 1970, when the race was on to find human retroviruses which cause cancer, there was a very good reason why clear, EM visualisation of such human oncoviruses was abandoned. The simple truth was that they were unable, then as now, to find high titres of cell-free retroviruses in fresh human blood or plasma.

It is worth stressing at this stage that in the entire 15 years of 'HIV'/'AIDS' research, no micrograph has ever been published purporting to show purified, densely packed 'HIV' particles, recovered from the fresh plasma of 'HIV positive' subjects. Indeed, no such picture exists of any so-called human retrovirus, not even HTLV-I, the alleged cause of adult T-cell leukaemia. That is not to say that micrographs of alleged 'HIV' have not been published, but they invariably came from cell cultures grown sometimes for weeks, in the total absence of an immune system (it is worth remembering that 'HIV infection' is most aften diagnosed on the detection of antibodies supposedly specific to 'HIV'),and involving co-culturing with known cancerous cell-lines such as H9 or CEM, and stimulated with mitogens, hydrocortisone and other chemical activators - the standard laboratory methods of reactivating latent viruses.

 

Figure 2. "Purified HIV-1 preparations are contaminated by cellular vesicles. Purified vesicles from H9 cells (a)..".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

 

There is no better way to show the degeneration into sloppy imprecision in science characterised by the 'AIDS' war than to compare de Harven's micrograph with Fig. 2, a micrograph published in 1997 of material banded at 1.16.gm/ml in a sucrose density gradient. Prior to its publication, by Gluschankof et al. (Virology, 230, pp 125-133, 1997), it was claimed that material banding at this level represented pure retrovirus. However, this study, and another in the same issue by Bess et al., finally came clean and admitted that all sorts of debris and extraneous matter banded at the retroviral density in the sucrose medium, principally cellular microvescicles, something that de Harven had observed even in the 1960's.

Whereas de Harven has to use three arrows to identify impurities in Fig.1, Gluschankof et al by total contrast have to use three arrows to identify three 'viral' dots, which may or may not be retroviruses, in their culture-derived rubbish tip in Fig. 2. This comparison strikingly illustrates the precision and superiority of de Harven's method of purification to currently used methods.

Undoubtedly, sedimentation in sucrose density gradients has its uses, particularly in confirming viral identity. Viruses have specific buoyant densities, and it is indeed known that purified retroviruses form a distinct sedimented band at 1.16 gm/ml, but it is a major error to suppose that all material contained in the gross supernatant from a cell culture which bands at that level is pure retrovirus, as Fig.2 makes clear. Moreover, in plasma samples containing more than one variety of active virus, as in PWA's with several, concurrently active viral infections, distinguishing them by their morphology under EM may be difficult, so subsequent buoyant density tests may be needed. Even the use of surrogate markers may be legitimate, but only when numerous laboratories have established that a large quantity of virus particles, of identical shape and size, are invariably present in diseased tissues and the virus has been proved beyond any doubt to be the cause of the disease. Then, and only then, can markers or proxies be trusted to indicate viraemia. However, 'HIV' fails all these tests.

If Ho and Wei are correct, it must be possible to pellet down fresh plasma from a person or persons who have had a recent 'high viral load' test result and clearly see tightly packed identical viral particles using EM, in a quantity consistent with the amount of virus indicated by the 'viral load' test. The application of the de Harven methodology will clearly demonstrate the presence or absence of actual rather than virtual particles, and do away with bogus mathematical models, inappropriate use of PCR, proxies, surrogates and all the other trappings of modern scientific obfuscation. If the virus particles cannot be seen, then they are just not there, whatever the test may claim. If it is claimed that there are indeed 'HIV' particles in the plasma, but too few to form a pellet, then there is obviously not enough virus in the sample to be pathogenically significant. Had 'AIDS' researchers been able to photograph high titres of cell-free 'HIV' in fresh plasma using the de Harven method during the last fifteen years, undoubtedly they would have done so and published their results, even if only to silence critics like Peter Duesberg. There are no such micrographs in the entire 'AIDS' literature.

When assessing how much virus - viable, enveloped, infectious virion units - should be visible from the amount of :

" 'HIV' RNA's" determined by a currently used 'viral load test', important scientific evidence shows that the 'proxy' viral RNA's represent very few actual potentially infectious particles. As Duesberg and Bialy state in the heavily censored version of their 'viral load theory' critique published in Nature (ibid):

The senior researcher [George Shaw] of the Wei et al paper has previously claimed that the method they used overestimates by at least 60,000 times the real titre of infectious HIV [Piatak et al, Science, 259, pp 1749-1754, 1993]. 100,000/60,000 is 1.7 infectious HIV's per ml ... Further, Ho and a different group of collaborators have just shown [Cao, et al New Eng. J. Med 332, pp 201-208, 1995] that more than 10,000 'plasma virions', detected by the branched-DNA amplification assay used in their Nature paper, correspond to less than one (!) infectious virus per ml . And infectious units, after all, are the only clinically relevant criteria for a viral pathogen. [my emphasis]

A transcript of a public question and answer session at the 1998 World AIDS Conference in Geneva shows the following exchange between Huw Christie and David Ho:

Huw: .....If people have a viral load of 200,000 per millilitre, it should be possible, shouldn't it, to demonstrate particles, viraemia? Why is it necessary to use a technique which is designed for amplification of numbers?

Ho: This indeed has been supported by other forms of assays, including assays that do not amplify the target that you're trying to measure. For example, using the branched chain DNA technology the same type of results have been generated, and compared head to head and published in numerous scientific papers.

David Ho is curiously silent about the findings of the paper he co-wrote with Cao et al cited above by Duesberg and Bialy.

Thus it may be seen that the 'viral load test' at best translates into a barely, if at all, detectable level of virus-like particles which can have little relevance in 'AIDS' pathogenicity; at worst it is a specious argument to pump asymptomatic people full of expensive drugs which may cause more harm than good in the long run.

CALLING THEIR BLUFF - £1000 challenge

So confident am I that no such EM evidence can be produced by adhering strictly to the de Harven methodology, I am prepared to offer the sum of £1000 to the first person to submit just such a micrograph, prepared under stringent laboratory conditions. These are:

1. Only plasma centrifuged from fresh whole blood may be used in the experiment. No material derived from cultured cells will be considered, to rule out 'viral particles' which may be merely cultural artefacts.

2. The donor blood/plasma must be taken from a person/persons with a recent 'high-viral load' test result, and evidence for the date and result of the test (the number of 'HIV'- RNA's alleged) must be submitted, obviously with the name of the person/persons deleted to preserve donor confidentiality.

3. The donor must not be in receipt of protease inhibitors, AZT or any antiviral drugs.

4. Only cold heparinised Ringer's solution may be used to dilute the plasma 1/1 ( i.e. 50%).

5. The diluted plasma shall be first filtered by aspiration-filtration, through a 0.6 millipore membrane. The resulting filtrate #1 will then be filtered again, this time using a 0.22 millipore membrane and filtrate #2 will be submitted to ultracentrifugation.

6. Centrifugation at 30,000 g for two hours will be used to prepare a pellet, likely to be extremely small. This pellet will be fixed with glutaraldehyde and osmium, then carefully detached and embedded in epoxy resins following routine EM procedures.

7. The electronmicrograph shall be at least 19,500 x magnification, and must resemble that published in Fig.1 of this article for particle size and shape, but with one notable and important variation. 'HIV' has been deemed to be a lentivirus, possessing a dense core of truncated conical shape. An ultrathin slice of randomly packed lentiviruses must inevitably show a number of particles bisected to show this core lengthwise, as well as end-on, with a resultant apparent mixture of round and 'rod-shaped' dense cores. Any micrograph which does not clearly show this feature will be deemed not to represent the lentivirus 'HIV'.

8. This challenge is open to any qualified scientists, or microbiology students/lab technicians with the necessary lab skills and facilities to carry out the work.

Photos of the required electronmicrograph(s) plus full details of the methodology, along with brief details of the senders' qualifications, must be sent, preferably with proof of date of postage, to me c/o the Continuum office at the address in the magazine. The first submission received which fulfils the above requirements according to qualified scientific scrutineers will be presented with £1000, in cash or by cheque, whichever is desired. There is no time limit, and the offer will remain open indefinitely.

Although not qualifying for the £1000, reports of failure to detect significant quantities of virus in the fresh plasma of 'HIV' positive donors, under the above conditions, would be most welcome, and considered for publication in Continuum. Medical and scientific journals are notoriously reluctant to publish reports which disprove a currently held paradigm, thereby preserving their role as the upholders of orthodoxy. Modern science doesn't seem to be about debate any more. However, I should be very interested to hear from anyone who has tried to establish a visual record of the viremia predicted by a 'high viral load' test result - and failed. Hitherto, the 'AIDS Dissidents' have had to restrict themselves to picking holes and spotting paradoxa in the orthodox 'AIDS' literature. Surely it cannot be beyond the wit and resources of people like the Reappraising AIDS group in California to carry out this experiment. Many distinguished scientists in that group, possessed of more than enough expertise, could easily carry out the lab work, if they could get access to a lab and the relevant donor plasma samples. Instead of commenting on the orthodoxy's work, why can't dissident scientists do some of their own? As de Harven says, when he wanted to do just this in his lab in Toronto in the mid '80's, his students threatened a walk-out. Short of barricading ourselves in a hijacked lab, can any reader come up with another way to kick virological ass?

Modern microbiologists and virologists have developed, and continue to develop, a bewildering array of techniques to aid them in pursuit of their disciplines. However, the increasing sophistication of the technology carries with it a proportionally increased need for scrutiny and analysis of their lab results. Modern culture techniques, involving mitogenic stimulation, other chemical additives, co-culturing with known cancerous cell-lines, the use of sucrose density gradients - all these valuable modern tools of science can easily produce results open to misinterpretation, accidental or deliberate. Add to this the pressure from financial interests typified by pharmaceutical companies seeking quick results to order, and pressure on labs to secure grant funding etc. and it is not difficult to see how a few 'virus-like particles', inevitably dredged up in cell cultures, can be parlayed into a massive viraemia by using 'proxies' and mathematical prestidigitation.

The uncritical acceptance of the gung-Ho 'viral load' theory has led to some patently risible studies. The latest absurdity comes from Farzadegan et al. in The Lancet (7.11.98), who carried out a long-term study based on blood samples taken from 650 male and female injecting drug users principally African Americans, using three different methods of measuring 'viral load'. The results of their trial claim to show that the women progress to 'AIDS' at the same rate as men but with only half the amount of 'viral load'. In other words, 'HIV' is supposedly twice as pathogenic in females as males - yet another pathogenic first for 'HIV'. At no stage do they mention the possibility that the harmful effects of the continuous use of illicit drugs far more logically explains the seeming equality of progression to AIDS in both sexes, irrespective of apparent differences in HIV kinetics between the sexes based on ambiguous 'viral load' tests. Unless their data are confirmed by similar studies of 'HIV positive' African, Asian and European males and females who are not IDU's, what does their study prove? Chillingly, the paper suggests that as the virus appears to be doubly pathogenic in women, they should be urgently considered for early drug cocktail therapy as soon as diagnosed.

In the final analysis, the only way to establish a true, in vivo viral titre in peripheral blood is by recovery of virus from a measured quantity of fresh, suspect plasma, and seeing the packed particles in a micrograph. Seeing, in this instance at least, is believing. As de Harven has explained (ibid), an aliquot of the unfixed viral pellet may be resuspended in Ringer's solution and used for titration by the precise, traditional method. Virus counting under EM may be tedious, but would, of course, reinforce the observations made. If few or no viral particles can be seen in the above conditions, then certain questions must be asked:

1. If not whole infectious viral particles, what is the 'viral load' test measuring?

2. What are the 'proxy' RNA's representing?

3. After administration of protease inhibitor drugs, and alleged decrease in 'proxy' RNA's, what has in fact been inhibited?

4. If little or no infectious virus is found in plasma of supposed viremic people, how did the haemophiliacs become infected by their plasma-derived clotting factors?

If, as I predict, no pathogenically significant amounts of virus can be visualised after pelleting down fresh plasma of donors previously deemed to be highly viremic by a 'viral load test', then perhaps we may be on the way to getting a re-examination of the whole concept of 'viral loads'; David Ho will have his Man of the Year award rescinded; the action of protease inhibitors can be reassessed in terms of a realistic risk-benefit ratio; and we can finally say "A pox on all your proxies!"

Acknowledgements: I should like to thank Dr. Etienne de Harven for his invaluable help in correcting and clarifying the technical aspects of the electron microscopy, and Alex Russell for diligent research, and Peter Duesberg for his usual kindness and common sense.

Continuum volume 5, number 5 - mid-winter 1999

 

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