Department of Medical Physics, Royal Perth Hospital, Western Australia,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso
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Where is the proof for HIV purification by any method?
In the rapid response “Re: The request remains the same and is still pure and simple” (12 June 2003), Brian Foley wrote: “Again I repeat "If the Perth Group would like to claim that density gradient ultracentrifugation is the ONLY acceptable method of isolating a virus, they should provide some evidence that any virus has ever been isolated by that method to their satisfaction, preferably another retrovirus”. Brian Foley does not appear to have read our rapid response “A simple request from the Perth Group” (5 June 2003) in which we wrote: “We do not care what method is used to isolate HIV. As far as we are concerned the only fact of importance is to obtain material which consists predominantly of particles with the morphology attributed to HIV and that the particles are infectious.”
Brian Foley also wrote “The Perth group is using a simple "straw man" argument, in claiming that their method is the only acceptable method of analysing a virus…”. The method of banding in density gradients is not our method. This is the method of: i. Barre-Sinoussi and Chermann, the principal and the 2nd author of the 1983 Montagnier et al paper who, in 1973, used it to purify the murine sarcoma virus and to obtain material which contained nothing else but particles with “No apparent differences in physical appearance” (1) ii. many other virologists who used it “to prepare highly purified concentrates of these relatively high yield viruses [murine, feline and avian retroviruses]. 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.” (2) iii. Bader, the renowned retrovirologist, according to whom “centrifugation to equilibrium in density gradients is the preferred technique for purification of RTV” (3) iv. Montagnier (4) and Gallo (5),(6) who used it to isolate / purify HIV and thus prove its existence.
Brian Foley wrote that the method of banding in density gradients: “has requirements included which make it IMPOSSIBLE to follow”. If this is the case then why has he accepted Montagnier’s and Gallo’s claim to have purified HIV by this method and thus to have proven the existence of the HIV genome and proteins, that is, the existence of a unique human retrovirus?
Brian Foley wrote: “It is well known that lentiviruses have mature and immature forms, with the electron dense core gradually forming as the viral particles mature. Thus any photograph of HIV or SIV or EIAV shows viral particles that are not all 100% identical”. It is true that in the culture supernatant there are mature and immature particles and thus the particles “are not all 100% identical”. However all the banded particles are mature which means that they should be identical.
Brian Foley wrote: “Density gradient ultracentrifugation is also rather harmful to viruses so that the viral particles loose much or all of their ability to infect cells after this treatment”. If this is the case then: i. why is banded material used to infect cultures? ii. why does Factor VIII contain infectious HIV particles when its manufacturing is even more taxing on retroviral particles than banding in density gradients?
Brian Foley wrote: “In addition to osmotic stress from the solutes used to generate the density gradient, there is also physical stress of ultracentrifugation which tends to remove viral envelope glycoprotein spikes”. This implies that the particles have spikes prior to banding. Although spikes are one of the main features of retroviral particles, so far nobody, not even Hans Gelderblom has published proof that such spikes are present on the cell-free “HIV” particles.
Brian Foley wrote: “If the Perth group believes that their method is valid for studying viruses, they should be able to name at least one virus which has been studied using their criteria. Where is the evidence that ANY virus has EVER been "purified" to their satisfaction??????” An example of purification is found in references (1) and (7).
(1) Sinoussi F, Mendiola L, Chermann JC, Jasmin C, Raynaud M. (1973) Purification and Partial Differentiation of the Particles of Murine Sarcoma Virus (M. MSV) According to their Sedimentation Rates in Sucrose Density Gradients. Spectra 4:237-243.
(2) Toplin I, Sottong P. (1972) Large-Volume Purification of Tumor Viruses by Use of Zonal Centrifuges. Applied Microbiology 23: 1010-1014.
(3) Bader, J.P. 1975. Reproduction of RNA Tumor Viruses, p.253-331. In:Comprehensive Virology Vol.4. H. Fraenkel-Conrat, R.R. Wagner (Eds.). Plenum Press, New York.
(4) Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioun C, Rozenbaum W, Montagnier L (1983) Isolation of a T-Lymphotrophic Retrovirus from a patient at Risk for Acquired Immune Deficiency Syndrome (AIDS). Science 220:868-871.
(5) Schupbach J, Popovic M, Gilden RV, Gonda, M. A., Sarngadharan, M. G., Gallo, R. C. (1984) Serological analysis of a Subgroup of Human T- Lymphotrophic Retroviruses (HTLV-III) Associated with AIDS. Science 224:503-505.
(6) Sarngadharan M, G., Popovic M, Bruch L. (1984). Antibodies Reactive to Human T-Lymphotrophic Retroviruses (HTLV-III) in the Serum of Patients with AIDS. Science 224:506-508.
(7) Crawford LV, Crawford EM. (1961) The Properties of Rous Sarcoma Virus Purified by Density Gradient Centrifugation. Virology 13:227-232.
Competing interests: None declared