Re: Re: Eight simple questions and a repeated request to Brian Foley 26 May 2004
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Christopher Tyler,
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Re: Re: Re: Eight simple questions and a repeated request to Brian Foley

Brian Foley wrote:
"You have been told numerous times that proving that a 1.16 g/ml band is 100% pure virus is not a requirement for studying retroviruses."
and;
"In some cases, poly-A RNA was extracted from density-enriched viral particles, and used for labeling probes for southern or northern blotting."

Does Brian Foley have proof that the 'density-enriched' material had even one single retroviral particle in it? If Gallo and Montagnier claimed they were 'purified' that implies there was AT THE VERY LEAST one retroviral-like particle in the density gradient. If they were so comfortable labeling these seminal density gradients 'purified' they MUST have had proof via electron microscopy that they had many more than one retroviral like particle IN THEIR DENSITY GRADIENTS. If so, why didn't they publish it?

Based on the language used by Brian Foley, '...was extracted from density-enriched viral particles', he also must have proof (not a belief) that they had particles in this 'enriched' material. Would he please provide those electron micrographs.


Brian Foley wrote:
"You repeatedly claim that it is, yet you have never shown anything but a rumor of a 1973 talk given by Francois Barre-Sinoussi and Jean Claude Bermann, to back up your claim. Do you have a transcript of that talk?"

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Toplin I. Tumor Virus Purification using Zonal Rotors. Spectra 1973;No. 4: 225-235.

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.

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In the first of the two papers from the Pasteur Institute meeting published in Spectra entitled "RNA tumor viruses purification using zonal rotors [RNA tumor viruses=retroviruses]", figure 1 is a "Flow chart for purification of RNA viruses by double sucrose density gradient zonal centrifugation". The flow chart is:


RNA Tumor Virus Purification:

VIRUS FLUID [80 litres]
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CLARIFICATION [4000G X 10 min.]
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K-3 ROTOR [RNAse-free sucrose 20-55% 12 litres/hour]
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K-3 VIRUS ZONE [500 ml., 30-38%]
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B-29 ROTOR [RNAase-free sucrose 30-45% 25,000 rpm X 180']
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B-29 VIRUS ZONE [150 ml., 32-37%]
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DIALYSIS OR ULTRAFILTRATION
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FINAL CONCENTRATE [150-200 ml.]


The particles thus obtained are then characterised by performing a number of assays. The flow chart for these assays is given in Table 3 and is as follows:


Assays for RNA Tumor Viruses:

Physical:

Electron Microscopy (neg stain and thin sect.)
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Virus count
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Morphology
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Purity


Biochemical:

Reverse transcriptase
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60-70S RNA, total RNA
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Total protein
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Gel analysis of viral and host proteins and nucleic acids


Immunological:

Gel diffusion
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Complement fixation
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Immunofluoroescence

(The latter two for with specific reagents for enveloped and internal antigens gs and env).


Biological:

Infectivity in vivo
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Infectivity in vitro

In the second paper "Purification and partial differentiation of the particles of murine [mouse] virus (M.MSV) according to their sedimentation rates in sucrose density gradients", Sinoussi, Chermann and their colleagues aimed to obtain a purified particle preparation and not to fully characterise the MSV. In double banding sucrose density gradients they obtained particles "banding in the region of the gradient corresponding to a density of 1.14-1.15 gm/ml". "No apparent differences in physical appearances could be discovered among the viral particles in these regions. There was no sign of aggregation of particles". They also showed that "The viral particles separated by zonal centrifugation are able to cause focus formation in murine embryonic fibroblast tissue cultures" and that reverse transcriptase (RT) "activity was found in the region of the gradient where particles were found".

Competing interests: None declared