Etienne de Harven, MD
"Le Mas Pitou"
2879 Route de Grasse
06530 Saint Cézaire sur Siagne
France
Tel. & Fax: (33) 4 93 60 28 39
E-mail: Pitou.Deharven@wanadoo.fr
I published the following picture in 1965 in a paper entitled Viremia
in Friend Leukemia: the electron microscope approach to the problem which
appeared in Pathologie-Biologie, vol 13, pp. 125-134. Transmission
electron microscopy was used to verify the success of a method for virus
purfication which I had developed when working at the Sloan Kettering Institute
in New York. The method was as follows: About 20 ml of blood from leukemic
DBA/2 mice was collected, blood cells were removed by low speed centrifugation
and the plasma was diluted 1/1 with cold heparinized Ringer's solution.
The diluted plasma was cleared from contaminating debris by two consecutive
steps of Millipore ultrafiltration, using pore size 0.65 µ first and 0.22
µ next. The second filtrate was then spun at high speed at 30000 g for
2 hours. The resulting pellet, about 1 mm in diameter, was immediately
fixed with osmium tetroxide, embedded in epoxy resin and prepared for electron
microscopy by routine thin sectioning methods. Aliquots of the unfixed
pellet were resuspended in Ringer's solution and used for titration of
the leukemogenic activity in adult DBA/2 mice, known to be 100% susceptible
to the virus. It. was that simple!
The picture shows, at a magnification of 19500 x, an almost pure population
of typical "type C" viruses (not yet called retrovirus in 1965....).
Three arrows point at contaminating debris and microvesicles. The interpretation
of these EM pictures was that virus purification was satisfactory and that
contamination rate was extremely low.
Dangerously enough, EM was progressively dismissed in retrovirus research
after 1970. Molecular biologists started to rely exclusively on various
"markers", and what was sedimenting in sucrose gradient at density
1.16 gm/ml was regarded as "pure virus". It is only in 1997, after
fifteen years of intensive HIV research, that elementary EM controls were
performed, with the disastrous results recently reviewed in Continuum.
How many wasted efforts, how many billions of research
dollars gone in smoke...
Horrible.
Errare humanum est sed diabolicum perseverare....
Etienne de Harven, MD
Member of The Group for the Reappraisal of the HIV/AIDS Hypothesis
Prof. Emerit. (Pathology) University of Toronto
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