OF AN ELECTRON MICROSCOPIST
by Etienne de Harven, MD
For an electron microscopist who spent most of his research career studying
retroviruses associated with murine leukemias and who followed closely
their hypothetical impact on the field of human oncology, it was predictable
that contemporary AIDS research was completely on the wrong track. The
following "Recollections" are presented to explain why.
The importance of electron microscopy in the emergence of modern cell
biology, between 1945 and 1965, is unanimously recognized. Unquestionably,
the relationships between cell structures and cell functions would never
have been elucidated without the high resolving power of the electron microscope
(EM). What is less generally recognized, however, is the role virus research
had in the study of cell ultrastructure. Historically, when Rüdenberg
filed the patent for the electron microscope in 1931 (1), his motivation
stemmed from the hope of visualizing the polio virus! And during the WW2
years, when electron microscopes started to be accessible to biologists,
attempts to visualize "virus particles" associated with cancer cells of
laboratory animals received first priority. Albert Claude, working at the
Rockefeller Institute, succeeded in demonstrating the Rous sarcoma virus
in chicken fibroblasts (2). And a few years later, Keith Porter et al.
had a similar success in imaging the "milk factor" in murine mammary adenocarcinomas
cells (3). The viral etiology of Rous sarcoma in chickens and of mammary
tumors in mice were well established by microbiological ultrafiltration
experiments, years before the EM pictures were published. Still, the direct
observation of virus particles in these experimental tumors gave an enormous
(today, we would perhaps say excessive!) impetus to virus research in oncology.
Viral etiology of several murine and avian malignancies had been clearly
demonstrated by ultrafiltration experiments which permitted the calculation
of the approximate diameter of the virus particles. Electron microscopists
knew, therefore, what size particle to look for, frequently around 100
nm. This facilitated the initial identification of oncogenic viruses by
EM, although it was later observed that many microvesicles or particulate
components of normal cells fall into a similar size range.
The discovery by Charlotte Friend, at the Sloan Kettering Institute
in New York, of a murine erythroleukemia transmissible by cell-free filtrates
illustrates well the research approaches around 1955. Moreover, since I
started working in Dr. Friend's laboratory at that time, the rationale
of our research program is clearly in my mind. For EM, we gave priority
to two types of sample: 1) a variety of tissues from leukemic Swiss mice
(spleen, lymph nodes, thymus and bone marrow), and 2) ultracentrifugation
pellets obtained from leukemic tissue cell-free filtrates which readily
transmitted the disease to adult Swiss and/or DBA/2 mice. We knew from
ultrafiltration experiments that the activity was absent when using filters
with an average pore size diameter smaller than 200 nm. Classic theories
on ultrafiltration indicated that the infective particles were therefore
probably in the 100 nm range. The study of thin sections from plastic embedded
leukemic tissue frequently revealed the presence of particles of approximately
that diameter, closely associated with a variety of cells. The particles
were bound by a single membrane and had a centrally located electron-dense
core or nucleoid. They had a characteristic structure and strikingly constant
diameters. To our knowledge, such particles did not resemble any known
components of normal cells. However, they did resemble particles previously
identified by others in several filterable experimental tumors and classified
by W. Bernhard as "type C" particles (4). More importantly, we observed
identical particles in ultracentrifugation pellets prepared from cell-free
filtrates capable of transmitting the disease to susceptible mice. On the
basis of these data, we hypothesized that these particles indeed represented
the oncogenic virus etiologically related to the Friend erythroleukemia
(5). Somewhat surprisingly, the virus was also found in close association
with cells not apparently related to the leukemic process, such as bone
marrow megakaryocytes, for example. These early EM studies also indicated
that all electron- dense particles averaging 100 nm in diameter are not
viruses and that a rigorous ultrastructural characterization was essential
for adequate differentiation between viruses and "virus-like particles."
Fortunately, our EM studies of Friend leukemia soon added an important
feature to the structural characterization of oncogenic RNA viruses. It
appeared that virus assembly is a cell surface phenomenon, the surface
membrane of the infected cell directly contributing the future viral envelope
by a multiple step mechanism for which we coined the word "budding" (6).
Viruses are released into intercellular spaces by such a budding process.
EM identification of viruses of that group in other experimental malignancies
became therefore more rigorous, observation of "budding particles" being
required. This probably helped in eliminating thousands of "virus-like
particles" observed in human malignancies and with which over-enthusiastic
electron microscopists attempted to contaminate the literature! In addition,
recognition of budding particles on cell surfaces clearly allowed us to
identify infected single cells, and to observe that such cells are perfectly
viable, with absolutely no ultrastructural evidence of any cytolytic effect
of viral infection. Moreover, typical viruses were frequently observed
in cells undergoing mitotic division (7).
Since obviously human experimentation was out of the question, the eventual
observation in human cancer cells of particles resembling those well characterized
in experimental tumors would have been of great interest, although by itself
not conclusive. Around 1960, many laboratories around the world, using
ever improving EM methodologies, were focusing on that target. At that
time, well before the emergence of molecular biology, EM was the best we
had to identify viruses in cell samples. The crucial role of EM was well
recognized at the Cold Spring Harbor conference of 1962, when Lwoff, Horne
and Tournier proposed classifying all viruses primarily on the basis of
their morphological characteristics demonstrated by electron microscopy
Continuing our studies on the Friend leukemia virus (FLV), and encouraged
by Dr. J. Beard from Duke University who had considerable experience with
avian leukosis, we oriented our effort toward the demonstration, by EM,
of viremia in leukemic mice. The most efficient initial step in purifying
avian leukosis viruses was to start not from leukemic tissues but from
the blood plasma of leukemic chickens. We wondered if a similar situation
could also prevail in leukemic mice. This was important for us because
the efficiency of our early virus purification method from homogenates
of leukemic tissues such as spleen or lymph nodes was poor. We developed,
therefore, a very simple purification procedure based on two steps of Millipore
filtration. A diluted plasma sample, around 10 ml (i.e. bleeding about
25 leukemic mice), was first clarified by aspiration filtration through
0.65 um filter; filtrate #1 was then further filtered through a 0.22 um
membrane; filtrate #2 was then centrifuged at 30,000g for 120 min. The
resulting pellet was extremely small...almost invisible, still worth embedding
for EM! Thin sections of such pellets revealed the presence of a most impressive
population of typical, well preserved virus particles, packed together,
and with very little contaminating debris (9). This was our approach to
the demonstration of viremia in 1965...
Meanwhile, many EM cancer research centers (those lead by Dr. W. Bernhard,
at Villejuif, France, Dr. A.J. Dalton, at NCI, Bethesda Md, Dr. L. Dmochowski,
at MD Anderson, Houston, TX, and us at Sloan Kettering, New York, NY),
were spending a considerable amount of time in attempts to demonstrate
virus particles associated with human cancer cells. "Virus-like particles"
were occasionally reported but convinced nobody. Typical viruses were never
conclusively demonstrated. This was in sharp contrast with the highly reproducible
demonstration, by EM, of viruses in a variety of murine and avian leukemias
and tumors. Very few papers were published to report on these negative
findings in human cancers and leukemias. However, Haguenau, in 1959 (10),
reported on the difficulty of identifying any typical virus particles in
a large series of human mammary carcinomas. Bernhard and Leplus, in 1964
(11) in an EM survey of cases of Hodgkin's disease, lymphosarcomes, lymphoid
leukemias and metastatic diseases failed to recognize virus particles associated
with these malignant conditions. At Sloan Kettering in New York, I decided,
in 1965, to stop surveying cases of leukemias and lymphomas by EM for the
presence of viruses in view of our entirely negative results. This was
reported at a conference on Methodological Approaches to the Study of Leukemias
held at the Wistar Institute, in 1965 (12).
Of mice and men
Publication of these negative findings failed to discourage fanatical
virus hunters! An explanation for these negative results had to be found
somewhere! Perhaps the technique of EM by the thin section method was not
the best approach? (although it worked perfectly for mice!). Preparing
thin sections was time-consuming and skill- demanding! Who had time for
that, when research funding was getting difficult, and when major pharmaceutical
corporations were starting to finance "crash programs" for speedy answers?...
Why don't we try the negative staining method? It is very easy and very
fast! And, after all, it gave beautiful results with unenveloped viruses
like adenovirus and polyoma. The results were absolute disaster because
fragile RNA tumor viruses (not yet called retroviruses...) are badly distorted
by air-drying during the negative staining procedures; they appear as particles
with a long tail! Unfortunately many cell debris and vesicular fragments,
when air-dried for negative staining, form similar "tailed" structures.
Interpreting "tailed" particles as RNA tumor viruses was therefore a bonanza
for virus hunters! Still, we had demonstrated that "tailed" virions were
preparation artifacts which can be avoided by proper control of the osmolality
and by osmium fixation prior to negative staining (13), or by critical-point
drying (14). The chaos created by reports on "tailed" particles damaged
the credibility of EM in the search for cancer related viruses. Cow's milk
and human milk were being screened for "tailed" particles and Sol Spiegelman
was eloquent on the possible risks of breast feeding...
A major discovery, which had nothing to do with EM, completely reoriented
ideas on how RNA tumor viruses might work, the discovery by Temin and Baltimore,
in 1970, of reverse transcriptase, RT. How RNA tumor viruses could induce
genomic alterations of the cells they infect suddenly became comprehensible.
Moreover, these viruses remained candidates as possible oncogens because
they were known NOT to be cytolytic. RNA tumor viruses were given a new
name, retroviruses, and the study of their eventual role in causing human
cancer soon received levels of federal funding, after Nixon's "War Against
Cancer Act," which were frighteningly in excess of what could have been
expected from a frequently recurring, perhaps interesting but so far totally
The profile of the research effort changed very drastically after the
discovery of RT in 1970. Somehow, most of the research methods which had
dominated the field of viral oncology from 1950 until 1970 were suddenly
substituted by the most exclusive fashion of molecular biology. I observed
this evolution almost as an outsider, since, in my view, electron microscopy
was no longer a primary contributing method to tackle any further the hypothetical
relationships between retroviruses and human cancer.
The 1970-1980 years were dominated by a series of ideas which would
never have withstood scientific scrutiny 10 or 20 years before. For example:
1. It became acceptable to postulate that when viruses cannot be seen
by EM in cancer cells, biochemical or immunological methods supposedly
identifying viral "markers" were enough to demonstrate viral infection
of the cells under scrutiny. Such markers can be an enzyme (RT), an antigen,
various proteins, or some RNA sequences. Never seeing the viral particles
was conveniently explained by the integration of the viral genome into
the chromosomes of the alleged infected cells. To accommodate such interpretations
implied complete oblivion of all we knew from previous research on cancer
of experimental animals. Admittedly, in these models EM was only showing
terminal steps of viral reduplication, initial phases being a series of
molecular events which escape ultrastructural recognition. Still, in all
the classical models such as murine and avian leukoses, visible terminal
steps of viral replication (i.e. "budding") were always observed and regarded
as essential for the spreading of infection from cell to cell.
2. Another shortcut with disastrous consequences has been the naive
notion that any material banding at 1.16gm/ml represented retroviruses!
Sure enough, true retroviruses band around that density. But this does
not mean that whatever material bands at 1.16gm/ml is retroviral in nature!
In the 1960s I was frequently asked to look at such 1.16gm/ml bands by
biochemists: "Look at this, it forms a sharp band, it should be pure viruses!"
Ultracentrifugation pellets obtained from such "sharp bands" showed, in
thin sections for EM, an extreme variety of microvesicles and proteinaceous
debris, but no retroviruses! Still, this approach has been (and still is!)
used to identify virus "markers!" How sad it is to think that a simple
EM control of such "bands" (which takes about two days, and costs a few
hundred dollars, but has never been done before 1997) could have prevented
these highly misleading interpretations of "markers" on which large budgets
have been simply wasted...
3. Collecting viruses from the supernatant of cultures of virus infected
cells raises other questions. We all remember the discovery, by Epstein
(15) in 1964 of the EB virus in cultures derived from African Burkitt's
lymphomas. This was an EM finding and the virus was immediately and properly
classified as a member of the herpes group. To identify this DNA virus
in cultured cells it was necessary to look into partially degenerating
cells because, most obviously, the virus had a marked cytolytic effect.
By complete contrast, retrovirus-carrying cells maintain excellent viability
and released viruses can be easily recovered in the culture supernatant
without the need to apply any lymphokine or growth factors stimulation
to the cultures.
4. As far as scientific policy is concerned, research on potentially
oncogenic viruses was dominated by the retrovirus hypothesis. Federal funding
took the same direction, amplified by the incredibly naive idea that success
was primarily a matter of money! Unusually large levels of federal support
resulted in the creation of a retrovirus research establishment. Large
numbers of research jobs were created in this venture. The intellectual
freedom to think along other avenues of cancer research was rapidly dwindling,
especially when major pharmaceutical companies started to offer tantalizing
contracts to support polarized retrovirus research... The top priority
was to demonstrate, at any cost, that retroviruses had something to do
with human cancer, an hypothesis, however, which didn't receive the slightest
support throughout the 1970s. Such a misdirected research effort would
have been relatively inconsequential as long as public health was not involved.
Unfortunately, the emergence of acquired immunodeficiency syndromes (AIDS)
in 1981 gave the retrovirus establishment an opportunity to transform what
could have been only an academic flop into a public health tragedy.
What happened after 1981 is so well known to all the readers of RA that
I hesitate to elaborate any further. The events which have lead to today's
crisis have been reviewed and analyzed most convincingly by Peter Duesberg
(16). I must say that I read Duesberg's book with great attention but basically
with no surprise, because the way research had been conducted on retroviruses
in the 1970s had so dangerously set the stage for "Impure Science" (17)...
Soon after the first cases of "Gay related immune deficiency" were described
by Michael Gottlieb it was obvious for all observers that Gallo and his
associates were going to jump on the new syndrome as a Godsent opportunity
to attempt to justify the lavish federal budgets they had consumed on retroviruses
over the past 10 years. In 1980, the scientific community was getting more
and more concerned about the absence of results in "The War against Cancer"
based on retrovirus hunting. The minor episode of HTLV 1 was not enough,
by far, to calm the fears of grossly misdirected federal research funds.
The fact that the syndrome, soon tactically renamed "AIDS", had nothing
to do with cancer was apparently of little embarrassment for Gallo. Frequent
association with Kaposi sarcoma helped to blur the difference in the eyes
of the public.
Dominated by the media, by special pressure groups and by the interests
of several pharmaceutical companies, the AIDS establishment efforts to
control the disease lost contact with open-minded, peer-reviewed medical
science since the unproven HIV/AIDS hypothesis received 100% of the research
funds while all other hypotheses were ignored. The general public and the
medical community were made to believe that the presence of circulating
antibodies is diagnostic of this disease, that Koch's postulates were outdated,
that 90% of all cases of an infectious disease can be observed in males,
that viremia can be measured by PCR enhancement of RNA fragments even when
viral particles are not demonstrable, etc., etc...
Most conveniently, it was totally forgotten that heroin addicts were
known for many decades to expose themselves to immuno- deficiencies, that
nitrite inhalants have multiple toxic effects, that the extreme toxicity
of AZT was known for over 20 years, that known retroviruses never have
any cytolytic effects, etc., etc...
And to ensure that the AIDS establishment could profitably continue
to flourish, research on any dissenting (i.e. non-HIV) hypothesis was carefully
prevented by tight control of research funding and by the extreme difficulty
of publishing anywhere any dissenting views... In the late 1980s, I was
considering adding to my research program in Toronto more EM observations
on samples from AIDS patients. Unfortunately, by that time the media and
the CDC had so perfectly orchestrated the panic of a plague-like epidemic
that I was quickly made to understand that my assistants would all transfer
out of the lab if I had insisted to activate such a program... The HIV
seropositivity test was still at that time regarded as providing reliable
diagnostic data. Since then, Papadopulos and the Australian team have demonstrated
that this is very far from the truth...(18).
Since my retirement in France, I take any opportunity to speak out,
as openly as I can, along the lines of this little note. I am proud to
be part of the "Group for the Re-appraising of the HIV-AIDS hypothesis"
and I sincerely hope that the combined activities of "The Group" will soon
contribute to a complete re-appraisal of the etiology of AIDS for the best
interest of patient's care, and for a revival of scientific integrity in
Copyright 1998 by Prof. Etienne de Harven, MD; "Le Mas Pitou," 2879
Route de Grasse, 06530 Saint Cézaire sur Siagne, FRANCE. <Pitou.Deharven@wanadoo.fr>
1. Rüdenberg R (1932). Elektronenmikroskop (Electron
microscope). Naturwissenschaften 20, 522.
2. Claude, A (1947-1948). Studies on cells: morphology,
chemical constitution, and distribution of biochemical functions. The Harvey
Lectures, Series XLIII, pp 121-164.
3. Prter KR & Thompson HP (1948). A particulate body
associated with epithelial cells cultured from mammary carcinoma of mice
of a milk factor strain. J. Exp. Med.,88:15-85.
4. Bernhard W (1960). The detection and study of tumor
viruses with the electron microscope. Cancer Res. 20:712-727.
5. De Harven E & Friend C. (1958). Electron microscope
study of a cell-free induced leukemia of the mouse: a preliminary report.
J. Biophys. Biochem. Cytol. 4:151-156.
6. De Harven E & Friend C (1960). Further electron
microscope studies of a mouse leukemia induced by cell-free filtrates.
J. Biophys. Biochem. Cytol. 7:747-752.
7. De Harven E. (1962). Ultrastructural studies on three
different types of mouse leukemia; a review. In "Tumors induced by viruses"pp.
183-206, Academic Press, Inc. New York.
8. Lwoff A, Horne R & Tournier, P (1962). Cold Spring
Harbor Symposium on Quantitative Biology 27:51.
9. Friend C & de Harven, E (1965). A new method for
purifying a murine leukemia virus. Fed. Proc. 24, N° 2. And: de Harven
E (1965). Viremia in Friend murine leukemia: the electron microscope approach
to the problem. Pathologie-Biologie 13 (3-4):125-134.
10. Haguenau F (1959). Le cancer du sein chez la femme.
Etude comparative au microscope électronique et au microscope optique.
Bull. Assoc. Franç. Etude du Cancer, 46:177-211.
11. Bernhard W & Leplus R (1964). In "Fine structure
of the normal and malignant human lymph node". Pergamon Press, ed., Oxford.
12. De Harven E (1965). Remarks on Viruse, Leukemia and
Electron Microscopy. In:"Methodological Approaches to the study of leukemias".
Defendi, V. edit.; The Wistar Institute Press, Philadelphia, publ., pp.
13. De Harven E & Friend C (1964). Structure of virus
particles partially purified from the blood of leukemic mice. Virology
14. De Harven E, Beju D, Evenson DP et al. (1973). Structure
of critical point dried oncornaviruses. Virology 55:535-540.
15. Epstein MA, Achong BG & Barr YM (1964). Virus
particles in cultured lymphoblasts from Burkitt's Lymphoma. Lancet 1:702-703.
16. Duesberg P (1996). "Inventing the AIDS Virus", Regnery
Publishing, Inc., Washington DC.
17. Epstein S (1996). "Impure Science; AIDS, Activism,
and the Politics of Knowledge". University of California Press, publ.,
18. Papadopulos-Eleopulos E, Turner VF & Papadimitriou
JM (1993). Is a positive Western blot proof of HIV infection? Bio/Technology