In his rapid response "For some people it’s April Fool’s day all
year round" (3
April 2003) with regards to the Padian
study, Peter Flegg wrote "I explicitly stated that "there was a low
transmission rate" in her study…". It must be pointed out that in
the prospective study of 175 HIV–discordant couples, tested every six months,
not one of the 175 non-infected men or women became HIV positive. That is, the
prospective Padian study did not prove a low transmission rate but a zero
transmission rate, that is, no transmission.
As far as Peter Flegg being perplexed about the thinking of
"dissidents", does he expect all scientists to think alike? Science
develops by a diversity of views and by questioning existing paradigms. While we
agree with the main views held by other "dissidents", that is, there
is no proof that HIV cause AIDS, there are also differences. We also agree with
some views held by the "HIV" experts including the following:
We agree a positive antibody test in individuals from the AIDS risk groups
indicates a propensity for the development of some diseases. However,
nowhere in the scientific literature is there evidence that a positive test
proves HIV infection. In other words, the antibody test is similar to the
erythrocyte sedimentation rate (ESR), a laboratory test of considerable
clinical utility that predicts the onset or presence of illness and whose
level is used to monitor the natural history or treatment of diseases.
Nonetheless, it is devoid of specificity.
Public Health Policy – like the "HIV" experts, we believe safe
sex is necessary for the prevention of AIDS. In fact we go one step further.
The HIV experts advise safe sex need be practised only with a positive
partner. We say safe sex should be practised with all partners irrespective
of antibody status.
Like the "HIV" experts, we are all for clean needles and again we
go one step further. Since, in our view it is the content of the syringe, that
is, the recreational drug injected which is one of the primary causes, the
best way to prevent AIDS is to avoid all injections.
Like the "HIV" experts we do not believe in conspiracy theories.
Peter Flegg also wrote we claim that HIV "spreads through anal
intercourse". In fact, in one of our rapid responses we wrote "It is
also our view that a positive antibody test ("HIV") is acquired by the
same means, that is, passive anal intercourse."( 24th
March 2003) In another rapid response we
wrote "all the presently available data prove it is only passive anal
intercourse which is the risk factor for the acquisition of a positive antibody
test." (1st
April 2003.) This means that either HIV
is the only unidirectionally spread STD in the history of medicine or whatever
causes the positive antibody test cannot be an infectious agent.
It is true that in some of our articles, to present our argument we have
assumed the existence of HIV in order to show how such an assumption leads to
untenable claims for the HIV theory of AIDS.
Peter Flegg wrote that we have been "asserting that HIV does not
exist". Peter Flegg may have interpreted our published material in this
manner but it is important to state that we have never said HIV does not exist.
Rather, from the very beginning when Montagnier and Gallo published their
studies in 1983/1984, we have consistently argued that neither their data nor
anybody else’s prove that HIV exists. (This issue can be resolved by
performing experiments. This is exactly what we argued for and what was accepted
by both sides at the Presidential AIDS Panel Meetings held in July 2000,
Johannesburg, South Africa http://aidsmyth.addr.com/panelreport.htm).
Some of the reasons can be found in what follows.
INTRODUCTION
Following the appearance of AIDS in 1981 many aetiological factors were
proposed. In May 1983 Montagnier announced the discovery of a retrovirus, now
known as HIV, from lymphatic tissue of a gay man with lymphadenopathy. One year
later Gallo reported data which "suggests that HTLV-III [HIV] is the
primary cause of AIDS". By 1986 the scientific community accepted the Gallo
assertion that the same data was "clearcut evidence" that HIV is the
causative agent of the clinical syndrome. Even today the five Science
papers published by these French and American groups are still widely regarded
as proving beyond all reasonable doubt that HIV exists and is the cause of AIDS.
However, not all scientists accepted these findings. In 1987 Peter Duesberg
published an invited paper in Cancer Research on retroviruses and cancer
in which he also questioned the role of HIV in AIDS. At the same time one of us
(EPE) also challenged the theory including the data claimed to prove the
existence of HIV. Papadopulos-Eleopulos also proposed an alternative,
non-infectious aetiology and treatments based on this hypothesis. Since then our
group has published papers addressing every facet of the HIV theory including a
detailed examination of HIV isolation and the HIV genome. Here we confine
ourselves largely to addressing the data published by Montagnier and Gallo in
their 1983/84 Science papers. Genomic data are only briefly discussed
because the existence of HIV and the HIV theory of AIDS were universally
accepted before such data were available. For a comprehensive discussion on
genomic data the reader is referred to reference 19.
RETROVIRUSES AND THEIR IDENTIFICATION
A virus possesses two characteristic properties. The first is anatomical,
that is, being a microscopic particle of individual morphology, the second, the
ability to generate identical progeny by synthetic processes obligatorily
occurring within living cells. It is the latter attribute which defines a
particle with the appearances of a virus, that is, a viral-like particle, as
infectious and thus a virus. The three subfamilies (0ncovirinae, Lentivirinae
and Spumavirinae) of Retroviridae (Retroviruses) are
"enveloped viruses with a diameter of 100-120 nm budding at cellular
membranes. Cell released virions contain condensed inner bodies (cores) and are
studded with projections (spikes, knobs)". The retroviral particles contain
RNA and the enzyme reverse transcriptase (RT), an RNA dependent DNA polymerase
which catalyses the synthesis of DNA contrary to the central dogma of biology,
that is, in a direction "reverse" from DNA to RNA. According to
retrovirologists, such DNA is then integrated into existing cellular DNA as a
"provirus". Retroviral particles share the property of concentrating
(banding) at a density of 1.16 gm/ml when centrifuged at high speeds in sucrose
density gradients, a fact long used in their purification.
All retrovirologists agree that to prove the existence of a new retrovirus
one must isolate it. However, the term "virus isolation" is beset with
semantic difficulties and ambiguities. The dictionary meaning of
"isolation" derives from the Latin insulatus (made into an
island) and refers to the act of separating an object from all other matter that
is not that object. "Purification" means to obtain something free from
impurities. In this context isolation is the same as purification. Because virus
particles are small it is not possible to obtain a single, isolated particle.
The next best thing is to obtain a mass of particles separate from everything
else. Until the early 1980s, for the isolation of animal retroviruses as well as
the "first" human retrovirus HL23V, by isolation retrovirologists
meant purification. On the other hand, nowadays both basic and specialised texts
rarely define "isolation" and when they do such attempts are
non-illuminating. For example, Levy defines isolation as a "sample of a
virus from a defined source", and White as the ability to "identify a
totally unforeseen virus, or even discover an entirely new agent".
Encompassed as "virus isolation" are listed methods of culturing
specimens in tissue and chick embryo cells, as well as live animals, following
by documentation therein of cytopathic and pathological effects, haemoabsorption,
immunofluorescence, antigen/antibody reactions and "characterisation of the
viral genome". HIV experts, including Luc Montagnier and Robin Weiss define
"virus isolation" as "propagating them [viruses] in cells in
culture" and see www.theperthgroup.com/aids/vftweiss.html.
However, if "virus isolation" is to "take a sample of a virus
from a defined source", or "propagating them in cells in
culture", then first one must have prior proof that a virus exists in
"a defined source" or "in cells in culture". One cannot know
that a virus exists or define its constituents without purification (isolation)
of the putative viral particles.
There are several reasons why this is mandatory:
To prove that the retrovirus-like particles are infectious, that is, they are
a virus
The finding of particles with the appearances of retroviruses, is not proof
that such particles are retroviruses and even less proof a particle is a
particular retrovirus. Particles bearing the morphological characteristics of
retroviruses are ubiquitous. In the 1970s such particles were frequently
observed in human leukaemia tissues, cultures of embryonic tissues and "in
the majority if not all, human placentas". Type-C retroviral particles are
present in "fish, snakes, worms, pheasant, quail, partridge, turkey,
tree-mouse and agouti" as well as in "tapeworms, insects...and
mammals". Gallo was well aware of this problem as far back as 1976 when he
wrote: "Release of virus-like particles morphologically and biochemically
resembling type-C virus but apparently lacking the ability to replicate have
been frequently observed from leukaemic tissue". In other words, it is not
possible to claim a particle is a retrovirus merely by appearances. To prove
that retrovirus-like particles observed in a culture are a virus one must
isolate the particles, characterise their proteins and RNA and introduce the
particles into a secondary culture. If any particles are released in the
secondary culture they too must be isolated and proven that their proteins and
RNA are the same as those from the primary culture. In such experiments one must
not ignore the use of legitimate controls and in doing so take in consideration
an important difference between retroviral and other infectious agents.
When one finds an infectious agent, for example a virus or a bacterium,
either in vitro or in vivo, one may be assured that the agent has
been introduced into the culture or animal from outside. Retroviruses are the
exception. This is because normal human and animal genomes contain information
which, under the appropriate conditions, leads to the synthesis of retroviral
RNA and proteins, or even to the assembly of retroviral particles, that is, to
the expression of endogenous retroviruses. And although as late as 1994 both
Gallo and Fauci taught "there are no known human endogenous
retroviruses", it is known that at least 1% of the human DNA is retroviral
DNA and that endogenous retroviruses are present "in all of us".
Furthermore, new endogenous retroviral genomes may arise from rearrangements of
existing retroviral genomes, cellular DNA or both, caused by many factors,
including pathogenic processes. The expression of endogenous retroviral genomes
may arise spontaneously and may be significantly accelerated and the yield
increased by conditions which induce cellular activation. According to the
eminent retrovirologist George Todaro, "the failure to isolate endogenous
viruses from certain species may reflect the limitations of in-vitro
cocultivation techniques". Endogenously produced retroviruses are
morphologically and biochemically indistinguishable from exogenous retroviruses.
Because of this, the finding of identical retrovirus in serially
"infected" cultures/cocultures is not proof that the cells are
infected with exogenous retrovirus. One method which may assist resolve but will
not prove whether cells acquire virus from the outside (exogenously acquired
retrovirus, infectious retrovirus) and have not assembled a retrovirus from
information already existing in normal cells (endogenous retrovirus), is to
conduct control cultures/cocultures in parallel with test cultures/cocultures.
The only difference between test and control cultures should be the introduction
of tissue assumed infected into the test cultures. In every other respect
control cultures must be dealt with identically. For example:
because detection of RT and retroviral genetic sequences, and release of
retroviral particles depends on the metabolic state of the cells, the
physiological state of the cells used in the control cultures should be as
close as possible to the test culture;
because the mere act of co-cocultivation may lead to release of endogenous
retroviral particles, if test cells are cocultured, so should the controls;
extracts even from normal, unstimulated cells when added to the cultures
may increase endogenous retroviral expression. Because of this, when host
cells are cultured with supernatant or material which bands at 1.16 gm/ml
from cultures thought to be infected, the controls must be cultured with
similar material from noninfected cultures;
since the appearance of endogenous retrovirus can be accelerated and the
yield increased a million fold by stimulating the cultures with mitogens,
mutagens, chemical carcinogens and radiation, if test cultures are exposed
to or employ such agents so should the controls;
to avoid observer bias and in the best interests of science, blind
examination of test and control cultures/cocultures should be performed.
To determine their biological effects
Without recourse to pure particles it is impossible to determine whether
effects are due to virus particles or contaminants including "chemical
stimulants", a caveat stressed as far back as 1911 by the Peyton Rous, the
father of retrovirology.
In 1911 Rous induced malignancy in chickens by injections of cell-free
filtrates obtained from a muscle tumour. Similar experiments were repeated by
many researchers and the tumour inducing filtrates became known as filterable
agents, filterable viruses, Rous agents, Rous virus and ultimately retroviruses.
However, Rous himself expressed doubts that the agents which caused the tumours
were infectious in nature. Indeed he warned, "The first tendency will be to
regard the self-perpetuating agent active in this sarcoma of the fowl as a
minute parasitic organism. Analogy with several infectious diseases of man and
the lower animals, caused by ultramicroscopic organisms, gives support to this
view of the findings, and at present work is being directed to its experimental
verification. But an agency of another sort is not out of the question. It is
conceivable that a chemical stimulant, elaborated by the neoplastic cells, might
cause the tumour in another host and bring about in consequence a further
production of the same stimulant".44
To characterise the viral proteins
The only way to prove that a protein is a constituent of an object is to
obtain it from that object, or when the object is very small as is the case of
viruses, from material consisting of purified virus particles. If the material
contains impurities which are proteins or contain proteins, it is not possible
to determine which are viral and which are not. Yet only after the viral
proteins are characterised is it possible to employ them as antigens in antibody
tests.
To characterise the viral genome
As for viral proteins the only way to prove that a stretch of RNA is viral it
is to obtain it from material which contains nothing else but virus particles.
If the material contains impurities the impurities must not include RNA. Then
and only then can the RNA and its complementary DNA (cDNA) be used as probes and
primers for genomic hybridisation and PCR studies.
To act as a gold standard for the antibody tests
The reaction of a virus or viral protein with an antibody present in a
patient's serum does not prove that the antibody is induced by or directed
against the virus or a viral protein. That is, it does not prove the reaction is
specific. This is because there are significant obstacles which hinder the
interpretation of antibody/antigen reactivity including non-specific
stimulation, cross-reactivity or both. Cross reactivity results from antibody
molecules, even monoclonal antibodies, interacting not only with the inducing
antigen but also with other antigens. Indeed, there are instances where
"cross-reactive antibodies may have higher affinity with antigens other
than the inducing antigen. Even antigens that differ for most of their structure
can share one determinant, and a monoclonal antibody recognising this site would
then give a 100% cross-reaction. An example is the reaction of autoantibodies in
lupus with both DNA and cardiolipin...It should be emphasised that sharing a
"determinant" does not mean that the antigens contain identical
chemical structures, but rather that they bear a chemical resemblance that may
not be well understood, for example, a distribution of surface charges".
Since polyclonal antibodies are composites of monoclonal antibodies these facts
apply equally, if not more so, to polyclonal antibodies. These facts have been
extensively exploited in clinical medicine for the diagnosis of diseases such as
syphilis and infectious mononucleosis. In these diseases, T. pallidum and
Epstein-Barr virus cause the appearance of antibodies reactive with ox-heart
proteins and sheep and horse red blood cells. However, this does not mean that
patients are "infected" with ox-heart, or horse red blood cells and
the diseases are induced by these agents. The only way to determine the
specificity of an antibody/antigen reaction is to use an independent method, a
gold standard to prove the presence or absence of the antigen. The only possible
gold standard for a test to prove a virus infection is the virus in question.
That is, virus isolation/purification.
METHODS FOR THE ISOLATION/PURIFICATION OF RETROVIRUS-LIKE PARTICLES
Up till the 1950s retroviruses were isolated/purified by filtration although
this method was less than satisfactory. With the development of the electron
microscope, apparently, for some retrovirologists, the detection of
retrovirus-like particles was deemed sufficient to prove the existence of a
retrovirus. However, other scientists including the well-known retrovirologist,
JW Beard, recognised that cells, including uninfected cells, under various
conditions, were responsible for the generation of a heterogenous array of
particles some with the appearances of retroviruses. Beard stressed:
"identification, characterisation, and analysis are subject to well-known
disciplines established by intensive investigations, and the possibilities have
by no means been exhausted. Strangely enough, it is in this field that the most
frequent shortcomings are seen. These are related at times to evasion of
disciplines or to their application to unsuitable materials. As was foreseen,
much of the interest in the more tedious aspects of particle isolation and
analysis has been diverted by the simpler and undoubtedly informative processes
of electron microscopy. While much can be learned quickly with the instrument, it
is nevertheless clear that the results obtained with it can never replace, and
all too often may obscure, the need for the critical fundamental analyses that
are dependent on access to homogenous materials" (italics ours).
By the 1970s there was general agreement that "Virions of RTV
[retroviruses] have a characteristic buoyant density, and centrifugation to
equilibrium in density gradients is the preferred technique for purification of
RTV". The method of banding in density gradients is not ideal either.
Substances other than retroviruses may band at the same density. This is why at
a meeting held at the Pasteur Institute in 1972, Francoise Barre-Sinoussi and
Jean-Claude Chermann stressed that to claim purification of retrovirus-like
particles using sucrose density gradients it is absolutely necessary to prove,
using the electron microscope, that the 1.16 gm/ml band contains nothing else
but particles with "no apparent differences in physical appearances".
THE PHENOMENA CLAIMED TO PROVE THE EXISTENCE OF HIV
In May 1983 Luc Montagnier, Francoise Barre-Sinoussi, Jean-Claude Chermann
and colleagues published a paper in Science entitled, "Isolation of
a T-Lymphotropic retrovirus from a patient at risk for Acquired Immune
Deficiency Syndrome (AIDS)". This is the paper which, since the resolution
of the polemics between Montagnier and Gallo concerning allegations of
misappropriation by the latter of the French virus sent to the US by the Pasteur
Institute, is accepted as being the study which proved the existence of HIV.
There it was shown that mitogen stimulated lymph node cell cultures from a gay
man (BRU) with lymphadenopathy were able to transcribe the synthetic RNA
primer-template An.dT15. From this data Montagnier and his colleagues
concluded that BRU’s lymph node cells were infected with a retrovirus. The
finding of the same activity in the supernatant of a coculture consisting of the
same cells with stimulated lymphocytes from a healthy individual was considered
proof for virus transmission as well as isolation. In another experiment
supernatants from the cocultures were added to two, three day old, stimulated
umbilical cord lymphocytes cultures. "Electron microscopy of the infected
umbilical cord lymphocytes showed characteristic immature particles with dense
crescent (C-type) budding at the plasma membrane". Supernatant from the
culture was banded in sucrose density gradient and the 1.16 gm/ml band was shown
to transcribe An.dT15. The proteins in the 1.16 gm/ml band as well as
the proteins of a cellular extract were separated according to their molecular
weight using "denaturing buffer and electrophoresed on 12.5 percent
polyacrylamide-SDS slab gel". When the strips were incubated with human
sera many proteins from the cellular extracts were found to react with serum
from BRU, another gay man as well as a "healthy donor". In the strips
containing the proteins from the 1.16 gm/ml band three proteins including a p25
(‘p’ for protein, 25 for its molecular weight in thousands) were found to
react. They also reported that the p25 did not react with antibodies to HTLV-I.
The material banding at 1.16 gm/ml was claimed to be "purified, labelled,
virus" although no electron microscopic data were presented. The authors
concluded: "A retrovirus belonging to the family of recently discovered
human T-cell leukaemia viruses (HTLV), but clearly distinct from each previous
isolate, has been isolated from a Caucasian patient with signs and symptoms that
often precede the acquired immune deficiency syndrome (AIDS). This virus is a
typical type-C RNA tumor virus, buds from the cell membrane, prefers magnesium
for reverse transcriptase activity, and has an internal antigen (p25) similar to
HTLV p24".
Robert Gallo and his associates did not consider the Montagnier group data as
proving "true isolation". As late as 1997, in a book published by one
of the best known HIV experts, Jaap Goudsmit, one reads: "The BRU lymph
node was first cultured in early January 1983 and, on January 15, it shed an
enzyme absolutely unique to the lentivirus group. [The enzyme is not even
specific to retroviruses much less to Lentiviruses (see below)]...The BRU
virus grew slowly and with difficulty, but its identity and activity were
reported in the May 20, 1983 issue of Science...The Pasteur Group was
widely acclaimed but very worried. In the world of virology, finding a new virus
is not enough: You must propagate and isolate the organism for analysis by other
virologists. The French had not yet isolated their new lentivirus".
Why then did: (a) Gallo, who reviewed the Montagnier manuscript, recommend
its publication? (b) all the HIV experts including Gallo and Goudsmit (on page
24 of his book one reads: "BRU was the first strain to be isolated")
accept that the first isolation of HIV and thus of its existence was proven in
the May 1983 Science paper?
A year later, in May 1984, Gallo, Popovic and their colleagues published four
papers in Science in which they claimed "isolation" of another
retrovirus from AIDS patients. However, in addition to the use of a leukaemic
cell line, the only difference between the Montagnier and Gallo groups’ data
were quantitative. In the first paper entitled "Detection, Isolation and
Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with
AIDS and pre-AIDS", (HTLV-III=HIV), experiments were described in which
"concentrated culture fluids harvested from short-term [mitogenically
stimulated] cultures of T-cells" from patients with AIDS or pre-AIDS were
cultured with a mitogenically stimulated leukaemic cell line HT and highly
selected clones obtained by culturing HT with irradiated cells of a healthy
donor. The data presented as proof of isolation of HIV were: (a) RT activity in
cell free supernatants and the 1.16 gm/ml band; (b) reaction in the cultures
with "Rabbit antiserum to HTLV-III" and "Patient serum (E.T.)";
(c) EM showing the presence of retroviral-like particles in the cultures.
An enquiry conducted by the National Institutes of Health Office of
Scientific Integrity found that the HT cell line was cultured not with
concentrated fluids (supernatant) originating from individual AIDS patients, but
with concentrated fluids pooled initially from individual cultures of three
patients and ultimately from the individual cultures of ten patients. In
evidence given to this enquiry the reason given was because none of the
supernatants "individually was producing high concentrations of reverse
transcriptase". In other words, Gallo and his colleagues did not regard the
levels of RT from individual cultures as proof that individual specimens
contained a retrovirus. The Gallo investigation found the pooling of specimens
to be "of dubious scientific rigor". One scientist described the
procedure as "really crazy". Most importantly how could Gallo use the
reaction with rabbit antiserum to prove HIV isolation (purification) when, to
obtain rabbit antiserum rabbits must first be injected with pure HIV? It is
inexplicable how Gallo and his colleagues could already possess "Rabbit
serum to HTLV-III" before they had proved the existence of a novel virus.
However, if the antiserum was manufactured "from rabbits infected
repeatedly with disrupted HTLV-III", that is, the material banding at 1.16
gm/ml, one would expect to obtain antibodies to all the proteins constituting
this material even if the proteins were cellular and not viral.
In the second paper entitled "Frequent Detection and Isolation of
Cytopathic Retroviruses (HTLV-III) from patients with AIDS and at risk of
AIDS", Gallo and his colleagues claimed to have "isolated" HTLV-III
(HIV) from 26/72 (36%) of AIDS patients. In this paper isolation was defined as
"more than one of the following": "repeated detection of a Mg2+-dependent
reverse transcriptase activity in supernatant fluids; virus observed by electron
microscopy (EM) [retrovirus-like particles in the culture]; intracellular
expression of virus-related antigens detected with antibodies from seropositive
donors or with rabbit antiserum to HTLV-III; or transmission of particles".
Transmission of particles was defined as "detected by RT assays or by
electron microscopic observation, to fresh human [umbilical] cord blood, bone
marrow, or peripheral blood T lymphocytes", cultured with supernatants from
the "infected" cultures. (It can be seen that the Gallo group method
permitted "isolation" of a retrovirus without evidence for either
particles or RT).
In the third paper, proteins from the 1.16 gm/ml band which they claimed was
"purified" HIV, as well as the proteins from the "infected"
cells, were "lysed and fractionated by electrophoresis on a 12%
polyacrylamide slab gel in the presence of SDS. The protein bands were
electrophoretically transferred to a nitrocellulose sheet" and reacted with
different sera. In other words Gallo used a technique which is known as Western
blot (WB) antibody test. Many proteins from the cellular extract were found to
react with sera from both patients and healthy individuals. They also reported
that two proteins from the 1.16 gm/ml band, p24 and p41, reacted with patient
sera. For this and no other reason it was claimed that "these molecules are
the major components of the virus preparation. p24 and p41 may therefore be
considered the viral structural proteins". As far as morphology is
concerned, the Gallo group reported that the HIV particle "is produced in
high numbers from infected cells by budding from the plasma membrane. A possible
unique feature of this virus is the cylindrical shaped core observed in many
mature virions...HTLV-III is a true member of the HTLV family". (HTLVs are
type C retroviral particles and not Lentiviruses as HIV is claimed to
be).
In the fourth paper, instead of separating the proteins which banded at 1.16
gm/ml and then incubating them with patient sera, the mixture of all proteins
was used, that is, they performed a test known as enzyme-linked immunosorbent
assay (ELISA). "To understand the molecular nature of the antigens
recognised by ELISA", they also performed WB, with some of the sera. They
reported that: "Serum samples from 88 percent of patients with AIDS and
from 79 percent of homosexual men with signs and symptoms that frequently
precede AIDS, but from less than 1 percent of heterosexual subjects, have
antibodies reactive against antigens of HTLV-III. The major immune reactivity
appears to be directed against p41, the presumed envelope antigen of the virus…The
data presented here and in the accompanying reports suggest that HTLV-III is the
primary cause of AIDS". Two years later Gallo wrote that "The results
presented in our four papers provided clearcut evidence that the aetiology of
AIDS and ARC was the new lymphotropic retrovirus, HTLV-III"67
(italics ours).
COMMENTS
As mentioned, apart from the quantitative difference, the Gallo group
experiments are no different from those performed by Montagnier and his
colleagues. It follows then, that if Montagnier's group data do not prove
"true isolation" neither did Gallo's nor anybody else’s because to
date everybody has repeated (for the vast majority only part thereof) the same
experiments as these two groups. It is of pivotal significance that neither
group reported the use of valid controls (see above) nor did they prove they had
obtained purified retrovirus-like particles. The question then is, do the data
obtained by the Montagnier and Gallo group, that is RT, particles in culture and
antigen/antibody reactions prove the isolation of a unique, or even a
retrovirus, any retrovirus?
Without doubt, if by isolation one expects proof of purification, then the
detection of an enzyme, or retrovirus-like particles in a culture, or proteins
either in the cells or the 1.16 gm/ml band which react with antibodies present
in human or animal sera, do not comply. To argue otherwise one must define the
detection of cardiac or hepatic enzymes in the blood of patients suffering chest
pain or jaundice as proof for isolation of the human heart or liver. Likewise,
if an antibody/antigen reaction is proof for isolation of a virus then antibody
reactivity to the protein βHCG is proof for isolation of the human
placenta. The assertion that the detection of a retrovirus-like particle in a
culture proves isolation is no different from asserting that the detection of a
fish-like creature in the ocean is the same as having a definite fish in your
frying pan. The detection of RT, retrovirus-like particles and antibody/antigen
reactivity can only be considered proof for the detection of a retrovirus, and
then if, and only if, there is prior knowledge that the three phenomena are
specific to retroviruses.
To claim that the detection of this phenomena proves the existence of a new
retrovirus one must have proof that at least one of the three phenomena is
different from that observed in all other known retroviruses.
Reverse transcriptase
At present some of the leading HIV researchers consider RT as being the
"sine qua non" of retroviruses and regard the detection of
reverse transcription in lymphocyte cultures from AIDS patients not only as
proof of the presence of such viruses but of HIV itself including HIV isolation
and quantification. However, according to some of the best known
retrovirologists including its discoverer, as well as the Nobel Laureate and
former Director of the US National Institute of Health Harold Varmus, reverse
transcriptases are present in all cells as well as bacteria and viruses.
"Reverse transcriptase (RT) was first discovered as an essential catalyst
in the biological cycle of retroviruses. However, in the past years, evidence
has accumulated showing that RTs are involved in a surprisingly large number of
RNA-mediated transcriptional events that include both viral and nonviral genetic
entities". Even if RT were a property only of viruses it is not specific to
retroviruses. According to Varmus, "Reverse transcription was assigned a
central role in the replication of other viruses [hepatitis B and cauliflower
mosaic viruses] and in the transposition and generation of other kinds of
eukaryotic DNA". "The hepatitis B viruses (HBVs) are small DNA viruses
that produce persistent hepatic infections in a variety of animal hosts and
replicate their DNA genomes via reverse transcription of an RNA intermediate.
All members of this family contain an open reading frame (ORF), "P"
(for pol), which is homologous to retroviral pol genes" [pol=polymerase].
"Hepatitis B virus (HBV) resembles retroviruses, including HIV, in several
respects. In particular, both viruses contain reverse transcriptase, and
replicate through an RNA intermediate". Because of this, it has been
suggested that hepatitis B infection should be treated with the same
antiretroviral agents as HIV infection.
At present, evidence exists which shows that although the major target organ
for hepatitis B virus is the liver, cells other than hepatocytes "including
peripheral blood lymphocytes and monocytes, may become infected with HBV".
Lymphocyte stimulation in general and PHA (an agent employed in the majority of
cultures with tissues from AIDS patients), is associated with the production of
hepatitis B virus from peripheral blood lymphocytes in patients infected with
HBV including "viral replication in chronic hepatitis B infection of
childhood". Hepatitis B virus infection is widespread in the AIDS risk
groups.
In the early 1970s Gallo proved that cultures of leukaemic cells transcribe
the An.dT15 template-primer as does material banding at 1.16 gm/ml
originating from "PHA stimulated (but not unstimulated normal human blood
lymphocytes". Reading the Gallo 1984 Science papers the impression
is gained that the leukaemic HT cell line and thus its clones, including H9
which Gallo used, was a new cell line developed in Gallo's laboratory. However
it is now known that the HT cell line is the HUT78 cell line which originated
from a patient with adult T cell leukaemia, a disease which Gallo claims is
caused by the retrovirus, HTLV-I. A year earlier Gallo claimed to have proven
that the HUT78 cells are infected with HTLV-I. If this is the case, the Gallo
group would have detected RT in their cultures even if the enzyme is specific to
retroviruses and the cultures were not infected with HIV. Other researchers
reported RT activity in normal non-infected spermatozoa.
It must also be pointed out that the presence of HIV reverse transcriptase
was detected by both Montagnier and Gallo, (and all HIV experts since)
indirectly, that is, by detecting the transcription of the template-primer An.dT15.
However, at least in 1984 if not 1983, Montagnier and his colleagues knew that
in the 1970s there was proof that "Among a number of template primers, (rA)n.(dT)12-18
has been most frequently employed since RT shows high activity with this
template primer. However, the problem is that the cellular DNA polymerases (pol b
and pol g ) also effectively utilize the same
template primer". In fact, in 1975, an International Conference on
eukaryotic DNA polymerases defined DNA polymerase γ as the cellular enzyme
which "copies An.dT15
with high efficiency but does not copy DNA well". One year earlier Gallo
wrote: "Under appropriate reaction conditions DNA polymerase β can
efficiently transcribe poly (A) primed by oligo (dT)". Thus it is possible
to detect transcription of An.dT15 even when no RT, either viral or
cellular, is present, especially under the conditions used to prove the
existence of HIV. (Both Montagnier and Gallo accept that the phenomena detected
in cultures which are said to prove HIV isolation cannot be detected unless the
cells are chemically stimulated). Nowadays the non-specificity of RT is
broadcast even in the popular press to readers contemplating the purchase of
shares in biotechnology companies.
In conclusion, even if one accepts Montagnier and Gallo's groups’
definition of isolation, given that RTs and reverse transcription are
nonspecific to retroviruses, their detection even in an unlimited number of
consecutive cultures/co-cultures cannot be considered proof for isolation and
propagation of a retrovirus.
"HIV" particles
Montagnier and his colleagues reported HIV initially as a type C particle,
then as a type D particle and then as a Lentivirus. In 1984 Gallo and his
colleagues reported HIV as a type C particle. However, in 1985 Gallo wrote:
"A possible unique feature of the virions is the cylindrical core observed
in many presumably mature virions. Virions having this type of core have been
frequently reported for certain type D retroviruses, and in some instances, for
type C retroviruses". Jay Levy reported HIV as a type D particle. Others at
the University of California wrote that "AIDS virus isolated show
morphologic characteristics of type C, type D and Lentiviruses". According
to Anthony Fauci and others" "T-cells and macrophages handle the virus
very differently. In the T-cell, virus buds out of the external plasma membrane
of the cell. In the monocyte/macrophage cultures it buds into membrane-bound
vesicles inside the cells". The latter is a description of a type A,
retroviral particle. Thus the leading HIV experts have described HIV as a member
of two subfamilies and three genera of Retroviridae. These taxonomical
differences imply that if HIV was a newly discovered mammal, it could have been
either human, a gorilla or an orang-utan. By consensus at present HIV is
regarded as a Lentivirus. This agreement was reached when it was realised
that in "HIV" positive individuals AIDS did not appear soon after
"infection", although the designation Lentivirus is a
morphological description.
"HIV infected cultures" contain in addition to the particles with
the morphology attributed to HIV many other "viral particles". For
example: Hockley and his colleagues from the Electron Microscopy and Photography
Section and Division of Virology at the National Institute for Biological
Standards and Control in the United Kingdom describe a profusion of particles
which they divide broadly into three groups, mature, ring-like and small with
spikes. The mature particles "were approximately spherical in shape and 100
to 150nm in diameter. The outer lipid membrane was frequently broken or absent
in places, and there was no evidence of surface spikes...A few mature particles
were found that were larger than average and appeared to contain a double
nucleoid...in the preparation of HIV there were always many vesicles with
granular contents in which it was not possible to recognise a distinct nucleoid".
Also, "The ring-like particles had a more consistently spherical shape and
were larger (140nm in diameter)" and the small particles "were
unusually spherical but sometimes slightly angular in shape and 65 to 90nm in
diameter" and had spike-like projections on their surface. Hans Gelderblom
who has done most of the EM studies in HIV/AIDS research reported that although Lentiviruses
and thus HIV is considered to have a cone shaped core, he and his colleagues
found centrosymmetric and tubular cores as well. The caption to one photograph
reads: "Virions can be seen having either elongated, 'baton-like' tubular
cores 30-35nm in diameter or containing more than one core. Tubular and regular
cone-shaped cores can coexist within one virion". The text states:
"Rarely, tubular core structures reminiscent of batons with a diameter of
30-35nm and a length of 150-250nm are observed". Lekatsas and other
virologists from Pretoria and Johannesburg reported: "We used the
characteristic cylindrical structure in the core as an identifying
characteristic for the virus to distinguish it from cellular debris and also
noted that it may vary considerably in its dimensions and morphological
features. We have found two basic virus particle sizes, 90nm and 120nm, both
present in large numbers. The larger particle bears no surface projections while
the smaller particle is rarely 'naked' and usually bears projections". The
US CDC reported: HIV particles are "usually round and have a diameter of
about 85-95nm...Virus with bar-shaped nucleoids and particles with a tear-drop
shape are commonly seen in HTLV-III/LAV infected lymphocytes, sometimes
ring-shaped particles without dense nucleoids are also seen". The question
then arises if the particles with the "unique" morphology considered
to be HIV represent an exogenous retrovirus originating from tissues of AIDS
patients or those at risk, then what is the origin and role of the many non-HIV
particles and which, if any, of the "HIV" or non-HIV particles band at
1.16 gm/ml? That is, which have the density characteristic of retroviruses?
Retrovirus-like particles have been found in non-HIV-infected cord blood
lymphocytes cell cultures and in cells used for HIV "isolation" such
as and H9 (HUT-78), CEM, C8166 and EBV transformed B-cells. Retrovirus-like
particles antigenically related to HIV have been found in cultures of salivary
gland extracts from patients with Sjorgen's syndrome. In the only EM study,
either in vivo or in vitro in which suitable controls were used
and in which extensive blind examination of controls and test material was
performed, particles indistinguishable from "HIV" were found in 18/20
(90%) of AIDS as well as in 13/15 (87%) of non-AIDS related lymph node
enlargements. This led the authors to conclude: "The presence of such
particles do not, by themselves indicate infection with HIV".
It is of pivotal significance to note that:
As Hans Gelderblom and his colleagues pointed out in 1998, to date nobody
has reported the presence of "infectious plasma HIV".
In no "HIV-infected" cultures are there particles which display
both principle morphological characteristics of retroviruses, that is,
"a diameter of 100-120nm" AND surfaces which "are studded
with projections (spikes, knobs)". The electron microscopist agree that
the "HIV" particles are devoid of knobs and thus of the
"HIV" protein gp120 (see below) said to be the constituent protein
of the knobs.
Hans Gelderblom and his colleagues have estimated that immediately after
being released from the cell membrane "HIV particles" possess an
average of 0.5 knobs per particle which are rapidly lost, but also pointed out
that "it was possible that structures resembling knobs might be observed
even when there was no gp120 [knobs] present, i.e. false positives".Yet
all HIV experts agree that the infectivity of the HIV particles is determined by
the gp120 (knobs). Thus, according to Montagnier et al, "The gp120
is responsible for binding the CD4 receptor" while for Matthews and
Bolognesi, "First gp120 binds to the CD4 receptor on an uninfected cell;
then gp41 becomes anchored in the adjoining membrane; next the two membranes
begin to fuse, and the virus spills its contents into the cell". Callebaut et
al state, "The human immunodeficiency virus (HIV) infects lymphocytes,
monocytes, and macrophages by binding to its principal receptor, the CD4
molecule, through the viral envelope glycoprotein gp120. The V3 loop of gp120 is
critical for HIV infection". Others are in complete agreement.
The general agreement that gp120 (knobs) is absolutely necessary for HIV
particles to be infectious and the fact that this protein (knobs) is not present
in the cell free particles leads to one unavoidable conclusion, that is, the
"HIV" particles are not infectious, they are not viral particles.
Furthermore,
To infect umbilical cord lymphocytes and the HUT-78 cells Montagnier and
Gallo used cell-free fluids (supernatants). Montagnier cultured umbilical
cord lymphocytes with supernatant from the cocultures of lymphocytes from
BRU cultured with lymphocytes from a healthy individual. Gallo cultured the
HUT-78 cell line with supernatants from the cultures of ten
"infected" individuals. Even if the supernatants contained
particles, being cell free the particles would be devoid of knobs (gp120),
that is, they would not have been infectious. This means that even if
Montagnier and Gallo had proof that their umbilical and HUT-78 cloned
cultures contained particles with all the morphological characteristics of
retroviruses, that in sucrose density gradients the particles banded at the
1.16 gm/ml band and the band contained nothing else but particles, the
particles could not have originated from their patients. Furthermore, since
the only samples which the Pasteur Institute gave to the Gallo laboratory
were cell free, one must question the basis upon which Gallo was accused of
misappropriation of the French virus.
It is generally accepted that haemophiliacs are infected with HIV by
contaminated factor VIII. However, to date nobody has reported the presence
of "HIV" particles in plasma. Even if such particles were present
the particles would be devoid of gp120. Since gp120 is "crucial to
HIV's ability to infect new cells" it is not possible for haemophiliacs
to be infected with HIV through factor VIII administration. There is another
fundamental reason why it is impossible for haemophiliacs to be infected
with "HIV" from "contaminated" factor VIII. According to
a publication from the CDC,112 "In order to obtain data on
the survival of HIV, laboratory studies have required the use of
artificially high concentrations of laboratory grown virus...the amount of
virus studied is not found in human specimens or any place else in
nature,...it does not spread or maintain infectiousness outside its host.
Although these unnatural concentrations of HIV can be kept alive under
precisely controlled and limited laboratory conditions, CDC studies have
shown that drying of even these high concentrations of HIV reduces the
number of infectious viruses by 90 to 99 percent within several hours. Since
the HIV concentrations used in laboratory studies are much higher than those
actually found in blood or other body specimens, drying of HIV-infected
human blood or other body fluids reduces the theoretical risk of
environmental transmission to that which has been observed--essentially
zero". Given that factor VIII is dispensed as a freeze-dried powder
which spends many weeks or months waiting use, it is incomprehensible that
the CDC and others continue to regard patients with haemophilia at risk for
HIV infection via contaminated factor VIII concentrates and enigmatic that
another explanation for "HIV" and AIDS in haemophiliacs has not
been sought.4
The only conclusion one can draw from the electron microscopy data is that
the reported particles are non-HIV or even retroviral specific which means that
the detection of such particles, be it in an unlimited number of consecutive
cultures/cocultures, is not proof for isolation, no matter how isolation is
defined.
Furthermore:
Even if reverse transcriptase activity and retrovirus-like particles are
specific to retroviruses, they are not specific for a unique retrovirus. The
only evidence both groups presented for the existence of a unique
retrovirus, HIV, is the antigen/antibody reaction, as acknowledged by both
Montagnier and Gallo.
It is accepted that the finding of a retrovirus in culture, especially
under the conditions used by Montagnier and Gallo is not proof that the
retrovirus is present in vivo. The only evidence which both Gallo and
Montagnier's groups presented for the existence of HIV in vivo was
the antigen/antibody reaction.
Even today the only evidence given as proof that HIV is the cause of AIDS
is a "correlation" between the antibody test and the appearance of
AIDS.
It is obvious that:
the Montagnier and Gallo interpretation of the antigen/antibody reaction
is crucial for the HIV hypothesis of AIDS and in fact for the existence of a
unique retrovirus, HIV;
if the interpretations are not correct, one would have no choice but to
question not only the HIV hypothesis of AIDS but also the existence of HIV.
"HIV" antibodies
It has already been mentioned that the mere interaction between an antigen
such as a protein or a virus and an antibody does not prove any more than a
chemical relationship. In all other aspects the reaction may be totally
nonspecific. To prove the specificity of the reaction one must employ an
alternative, independent method of proving the existence of the antigen, that
is, one must use what is generally known as a gold standard. The only possible
gold standard for the HIV antibody tests (the antigen/antibody reaction) is HIV
itself, that is, HIV isolation. No matter how one defines isolation, obviously
the definition cannot include the antibody/antigen reaction (the antibody test,
neither the WB nor the ELISA) because, by doing so, not only does one not have
an independent analysis, the test becomes its own gold standard and is thus
rendered meaningless. Reading the 1984 Gallo Science papers it appears
that Gallo always defined isolation as "more than one of the following:
reverse transcriptase activity either in the supernatant or the 1.16 gm/ml band;
retrovirus-like particles in the culture or reaction between proteins (either in
the cultured cells or the 1.16 gm/ml band) with antibodies present in the
patients sera or "antiserum to HTLV-III". However, in an interview
Gallo gave to Huw Christie, the editor of the British magazine Continuum,
at the 1998 Geneva AIDS Conference, Gallo said, "Sometimes we had Western
Blot positive but we couldn't isolate the virus. So we got worried and felt we
were getting false positives sometimes so we added the Western Blot. That's all
I can tell you. It was an experimental tool when we added it and for us it
worked well because we could isolate the virus when we did it". In other
words:
In 1984 Gallo knew that to prove the specificity of the antibody test one
must use a gold standard.
The gold standard had to be HIV isolation.
Although it is not known how he and his colleagues initially defined
isolation, the definition did not include the antibody/antigen reaction (the
Western Blot). This means that Gallo and his colleagues were aware that an
antibody/antigen reaction cannot be used as proof for isolation. Yet, by the
time their Science papers were published, and in order to reconcile
the low correlation between what they initially called isolation and the
antibody/antigen reaction, arbitrarily, and against all scientific
reasoning, they "added the Western Blot" to their definition of
isolation. (It is interesting that even with their novel definition the
correlation between "isolation" and antibody tests was still less
than perfect. They "isolated" HIV from 26/72 (36%) of patients
with AIDS, while 88% of patients with AIDS were seropositive using an
antibody test Gallo considered highly specific).
In an interview which Montagnier gave to the French Journalist Djamel Tahi in
1997, he stated: "analysis of the proteins of the virus demands mass
production and purification. It is necessary to do that". Indeed, the only
way to prove that a protein is a viral constituent is to obtain it from material
which contains nothing else but viral particles. Instead Montagnier’s and
Gallo's groups incubated the proteins which banded at 1.16 gm/ml with sera from
AIDS patients. The proteins which were found to react more often with the sera
were said to be HIV proteins (although neither group published any evidence for
the existence of retrovirus-like particles, pure or impure, at the 1.16 gm/ml
band); and the antibodies to be HIV antibodies. However, even if the
antigen/antibody reaction is 100% specific, that is, antibodies react only with
inducing antigens and with no other, from such a reaction it is impossible to
determine the origin even of one reactant much less the origin of both the
antigen and the antibody. Let us consider differing scenarios:
Both Gallo and Montagnier had proof that the 1.16 gm/ml band contained
nothing else but retrovirus particles and that their antibodies reacted
specifically with their proteins. According to Montagnier, in the cultures
which contain cells originating from patients with adult T4 cell leukaemia,
as did Gallo's HUT-78 cell line, "it is a real soup" of
retroviruses. Indeed, even if the cultures do not harbour HIV they will
harbour HTLV-I and, given the condition used by Gallo and the fact that the
cells were leukaemic, they may also contain endogenous retroviruses.
According to one well known HIV expert, Myron Essex, 35% of AIDS patients
possess antibodies to HTLV-I. (In the same issue of Science in which
Montagnier published his isolation of "HIV", Gallo published three
papers claiming isolation of HTLV-I from AIDS patients suggesting this
retrovirus was the cause of AIDS). According to another equally as well
known HIV expert, Reinhard Kurth, from the Paul-Ehrich Institute in Germany,
70% of "HIV-positive patients" have antibodies which react with
the retrovirus HTDV/HERV-K, an endogenous retrovirus, or, as Kurth put it, a
retrovirus present "in all of us". Thirty seven per cent of HIV
positive individuals were also found to have antibodies to type D
retroviruses whereas HIV is claimed to be a Lentivirus. Although
Montagnier's cultures may not have contained HTLV-I, the result may still
have been "a real soup" of endogenous retroviruses especially if
one considers their culture conditions and the cells used, umbilical cord
lymphocytes, which have been shown to release retrovirus-like particles even
when not infected with HIV. Since we cannot obtain the proteins from each
particle and characterise them, the next best thing is to take the mass of
particles, disrupt their proteins and position them in an electrophoretic
strip according to their molecular weights. Although we know that the
proteins in the strip are retroviral, we have no way of determining which
protein belongs to which retrovirus if more than one retrovirus is present
at the 1.16 gm/ml band. When the proteins are incubated with sera, we may
find some of the proteins react. From such a reaction we will be able to say
that the antibodies and obviously the proteins are viral but we cannot
determine which protein belongs to which virus and by which virus the
antibodies were induced. We will be definitely wrong if we consider such a
reaction proof that the 1.16 gm/ml band: (a) contains only one retrovirus;
(b) the retrovirus is a new exogenous retrovirus, HIV; (c) the proteins are
the HIV proteins; (d) the antibodies are HIV antibodies; (e) these data are
proof that the patient is infected with HIV even if the antibody antigen
reactions are specific.
Both Montagnier and Gallo had proof that the vast majority of the 1.16
gm/ml band was composed of retrovirus particles but that the band also
contained non-retroviral material. This material could have been of cellular
origin (cellular constituents also band at 1.16 gm/ml) and may be of
bacterial, fungal and viral origin (constituents of the many infectious
agents other than retroviruses, known to be present in the cultures and the
patients). It is a fact that in the USA, Europe and Australia individuals
with AIDS as well as those at risk have antibodies to many infectious agents
including viruses such as EBV, CMV and hepatitis B virus. Evidence also
exists which shows that individuals with AIDS and those at risk have
circulatory immune complexes, rheumatoid factor, anti-nuclear,
anti-cellular, anti-platelet, anti-red cells, anti-actin, anti-tubulin, and
anti-myosin antibodies. Montagnier himself demonstrated that individuals
with AIDS and those at risk have high levels of antibodies to the ubiquitous
cellular protein actin whose molecular weight is 41,000, as well as to
another ubiquitous protein, myosin, which has two sub-units of molecular
weights, 18,000 and 25,000. Anti-lymphocyte antibodies have been found in
87% of patients who have a positive "HIV" antibody test and their
levels correlate with clinical status. It is also acknowledged that Africans
with AIDS and those at risk are infected with many agents other than HIV.
We know that it is not possible to take a protein from the 1.16 gm/ml mass
and know from which component it originates. Let us then follow the same steps
as Gallo and Montagnier. Take the mass of material banding at the 1.16 gm/ml,
disrupt the proteins and electrophoretically position the proteins in a strip
according to their molecular weights. Using this technique we are unable to
state from which component of the 1.16 gm/ml band mass a protein on the strip
derives. Next we incubate the proteins in the strip with patient sera and
discover that some of the proteins react with antibodies present in the sera.
Even if the antibody/antigen interaction is 100% specific such a reaction does
permit us to define the origin of the proteins. Yet from such a reaction and
without any proof that the antibodies specifically reacted, Montagnier and Gallo
defined the origin of both the proteins and the antibodies as "HIV".
As mentioned, Montagnier found three proteins in the 1.16 gm/ml band which
reacted with antibodies present in his patients’ sera. These were p25, p80 and
p45. Montagnier concluded that his patients were infected with a retrovirus
which "contains a major p25 protein, similar in size to that of HTLV-I",
but made no comment in regard to the p80 protein. Regarding p45 he wrote:
"The 45K [p45] protein may be due to contamination of the virus by cellular
actin".
In 1997 Montagnier said that the protein he detected in 1983 had a molecular
weight of 43,000 and was actin. (The molecular weight of actin is neither 45,000
nor 43,000 but 41,000. However since Montagnier and Gallo determined the
molecular weights of the proteins by their migration in an electrophoretic
strip, and because the migration may be influenced by other factors, for
example, by the protein's charge, it is possible that these slight differences
in the molecular weight are simply the result of experimental variation).
When Gallo and his colleagues used the cellular proteins as antigens in the
WB they reported: "The most prominent reactions were the antigens of the
following molecular weights: 65,000, 60,000, 55,000, 41,000 and 24,000. Antigens
with molecular weights of approximately 88,000, 80,000, 39,000, 32,000, 28,000
and 21,000 gave less prominent reactions…A large protein with a molecular
weight of approximately 130,000 and a protein of 48,000 were also
detected". In another experiment the "antigens from virus purified
from the culture fluids", that is, the 1.16 gm/ml band, were incubated with
different sera. They found an "Extensive" reaction of the AIDS
patients sera with p24 and p41 and concluded: "these molecules are the
major component of the virus-preparation. P24 and p41 may therefore be
considered viral structural proteins…Furthermore, an antigen with a molecular
weight of approximately 110,000 was detected in the virus preparation but was
below limit of detection in the cells. Also, p39 was present in the virus
preparation…Occasionally an additional set of antigens was recognised by a
serum but their relation to the antigens described above is unclear".
Between 1983-87 the detection of antibodies in patient sera which reacted
with p24 or p41 (Montagnier considered reaction with p24 and Gallo with p41 HIV
specific) was considered proof that the patient was infected with HIV. In the
same period of time it became obvious that a significant number of individuals
at no risk of AIDS had antibodies which reacted with these proteins. Since 1987
most of the proteins which Gallo found to react either in the cell extracts or
the 1.16 gm/ml band are now considered HIV proteins and laboratories require the
presence of antibodies which react with more than one protein before the patient
is considered infected with HIV. The number and identity of antibody/protein
(Western blot) bands required vary from continent to continent, from country to
country and even between and within laboratories in the same country. Thus it is
possible for the same patient to be HIV seropositive in New York for example,
but not in Africa or Australia.
Furthermore, for many years evidence exists which shows that p24 is not
"HIV" specific123,124, p41 is the cellular protein actin,58,125,126
p32 is class II histocompatability DR protein126,127 and p120 / p160
are oligomers of p41.128
Even if proof exists that the "HIV" proteins do indeed belong to a
unique exogenous retrovirus, it cannot be assumed that antibodies that react
with them are diagnostic of "HIV" infection. To prove the specificity
of the antibody tests it is mandatory to:
test a large number of subjects with and without AIDS. The subjects
without AIDS must not exclusively be healthy individuals (since they do not
have high levels of antibodies, one would expect few if any reactions) but
include the sick, such as patients with infectious diseases (other than
those which are said to result from HIV infection), those receiving
chemotherapy and those with auto-immune disorders;
simultaneously (preferably on the same blood sample) perform tests for HIV
isolation;
compare the antibody test results with the results of HIV isolation, that
is, use HIV as a gold standard for the antibody test.
To date nobody has published such studies using any definition of
"HIV" isolation.
The inescapable conclusion is that the antibody/antigen reaction is not HIV
specific and thus cannot be used to prove HIV isolation, no matter how isolation
is defined.
MORE RECENT DEVELOPMENTS
By 1997 some of the best known HIV experts accepted that:
"Purified" HIV contains cellular proteins, "some are
over-represented in comparison to the relative amount in the cell membrane,
whereas others appear to be absent", and that these proteins
"serve as protective immunogens in vaccination experiments".
HIV "used for biochemical and serological analyses or as immunogens
is frequently prepared by centrifugation through sucrose gradients",
but in none of the studies "the purity of the virus preparation has
been verified". In other words, up till 1997 nobody has published
electron micrographs of the 1.16 gm/ml band to prove that the "purified
virus" contained nothing else but virus particles.
In that year, in Virology, two studies were published, one by a USA
team, principal author Julian Bess, and the other by a Franco-German group,
principal author Pablo Gluschankof, with the first electron micrographs of
"purified HIV". While in the Gluschankof et al studies the EMs
were from the 1.16 gm/ml band, in the Bess et al band they were from
pooled bands. The authors of both studies claimed that their
"purified" material contained some particles with the appearances of
retroviruses and in fact were HIV particles. But they admitted that their
material predominantly contained particles which were not retroviruses but
"budding membrane particles frequently called microvesicles" or
"mock virus". Indeed the caption to the Gluschankof et al
electron micrograph reads, "Purified vesicles from infected H9 cells (a)
and activated PBMC (b) supernatants", not purified HIV. In further
experiments the supernatant from non-infected cultures was also banded in
sucrose gradients. Both groups claimed that the banded material from these
cultures contained only microvesicles, "mock virus" particles, but no
HIV. Both the "HIV" particles and the mock-virus particles possessed
membranes. In the USA study the "HIV-1 particles" were differentiated
from the microvesicles "by the electron dense cores", whereas in the
other study the "HIV" particles were "identified by the
relatively homogenous diameter of about 110 nm, the dense cone-shaped core, and
the "lateral bodies". However, in the arrowed particles which are said
to be HIV it is difficult if not impossible to locate any which have cone-shaped
cores or bilateral, "lateral bodies". In fact no particle in any study
has the principle morphological characteristic of retrovirus, a diameter of
100-120 nM and surface spikes, knobs. In the Franco-German study the average
"HIV" particle diameter is 136 nM and no particle had a diameter less
than 120 nM. In the USA study the corresponding dimensions are 236 nM and 160 nM.
In other words, the American "HIV" is twice the diameter of the
European "HIV", and all other "HIV" particles. The US
authors did not note or explain this discrepancy and "were much more
focused on showing the mixture of particles in the preparations as opposed to
their actual diameters". The diameter of the microvesicles "range in
size from about 50 to 500 nm". Both the "HIV" and the
"mock" virus particles contained RNA. The RNA of the latter had
contained mRNA which is known to be rich in adenine and which, according to
Gallo is specific to retroviral RNA. According to Gluschankof et al,
"The vesicles contain large amounts of protein and nucleic acids which are
unstructured and thus are transparent by electron microscopy", that is why
many, but not all, appear "empty" by electron microscopy, while the
nucleic acids in the "HIV" particles are structured and for this
reason they appear to have an "electron dense cores". However,
according to a leading retrovirologist, John Bader, the core density can be
changed by external conditions, that is, the culture conditions. It is well
known that a structural virus particle or virus-like particle can become
"unstructured" in the presence of reducing agents. The possibility
cannot be excluded then, that the apparent morphological differences between the
two types of particles may be due to nothing more than the difference in the
redox of the microenvironment in which they are assembled, released or both. It
is significant that actin polymerisation (or actin/myosin interaction)
"mediates HIV budding" and release. Evidence also exists that:
As shown by Bess et al, uninfected cells exhibit buds which are no
different from those in infected cells.
There is an association between the redistribution of polymerised actin,
myosin and other cellular proteins (glycoproteins) and many cellular
processes including budding unrelated to release of retrovirus.
Polymerisation of actin, actin-myosin interaction and cross-linking of
polymers in general is regulated by the redox state, oxidation leading to
interaction.
Both AIDS patients and cultures derived from AIDS patients are subjected
to oxidising agents. In fact, for the detection of "HIV" proteins
and particles the cell cultures must be stimulated (treated with oxidising
agents).
In the presence of antioxidants no "HIV" phenomena can be
observed.
The minimum absolutely necessary but not sufficient condition to claim that
what are called "HIV-1 particles" are a retrovirus and not cellular
microvesicles is to show that the sucrose density fractions obtained from the
"infected" cells contain proteins which are not present in the same
fractions obtained from non-infected cells. However, Bess et al have
shown this is not the case. The only difference one can see in their
SDS-polyacrylamide gel electrophoresis strips of "purified virus" and
"mock virus" is quantitative, not qualitative. This quantitative
difference may be due to many reasons including the fact that there were
significant differences in the history and the mode of preparation of the
non-infected and "infected" H9 cell cultures, in addition to the
"infection".
A similar finding was reported by the same authors a few years earlier.
However, while in both studies the proteins of molecular weight "near 42
kDa" (42,000) are labelled as "Actin" and "in the 30- to
40-kDa range" as "HLA DR", all the proteins with molecular weight
higher than approximately 42,000 and lower than approximately 30,000 are left
unlabelled in the earlier paper. In the 1997 study, three proteins of molecular
weight lower than 30,000 are labelled as p24CA, p17MA, and
p6/p7NC and are said to represent "major bands of viral
proteins". However, also according to the authors, "these labels were
added when one of the reviewers asked for them. He felt it would help orient
readers when looking at the figure - the reviewer is correct. We did not
determine the identities of the bands in this particular gel".
In their earlier study the researchers from the USA presented further
evidence that the "viral proteins" were nothing more than cellular
proteins. In their efforts to make an HIV vaccine they immunised macaques with,
amongst other antigens, "mock virus", that is, sucrose density banded
material from the supernatants of non-infected H9 cell cultures. After the
initial immunisation the monkeys were given boosters at 4, 8 and 12 weeks. The
animals were then challenged with "SIV" propagated either in H9 cells
or macaque cells. When the WBs obtained after immunisation but before "SIV"
challenge were compared with the WBs post-challenge, it was found that challenge
with "SIV" propagated in macaque cells had some additional bands.
However, the WBs obtained after the challenge with SIV propagated in H9 cells
were identical with the WBs obtained after immunisation but before challenge. In
other words, the protein immunogens in the "virus" were identical with
the immunogens in the "mock virus". Since both the "mock
virus" and "purified" virus contain the same proteins, then all
the particles seen in the banded materials including what the authors of the
1997 Virology papers call "HIV" particles must be cellular
vesicles. Since there is no proof that the banded, "purified virus",
material contains retrovirus proteins and thus retrovirus particles then there
can be no proof that any of the banded RNA is retroviral RNA. When such RNA (or
its cDNA) is used as probes and primers for hybridisation and PCR studies, no
matter what results are obtained, they cannot be considered proof for infection
with a retrovirus, any retrovirus.
In the interview which Montagnier gave to Djamel Tahi he was asked why they
did not publish an electron micrograph of the 1.16 gm/ml band to prove that the
band represented "purified" virus, as they claimed. He replied that
the reason for this was that even after "Roman effort" in their
"purified" virus they could not see any particles with the
"morphology typical of retroviruses. They were very different. Relatively
different". When Montagnier was asked if Gallo isolated HIV he replied:
"I do not believe so". If there were no retrovirus-like particles in
Montagnier’s "purified" virus, then obviously Montagnier and his
colleagues could not claim to have isolated a specific exogenous retrovirus,
HIV.
THE "HIV GENOME"
To claim that the stretch of RNA (cDNA) is the genome of a unique retroviral
particle, HIV, the most basic requirement is proof for the existence of a unique
molecular entity "HIV RNA" ("HIV DNA") that is, a unique
fragment of RNA (DNA) identical in both composition and length in all infected
individuals. The claim that a stretch of RNA (cDNA) is a unique molecular entity
which constitutes the genome of a unique retrovirus can be accepted if and only
if it is shown that the RNA belongs to particles with the morphological,
physical and replicative characteristic of retroviral particles. Proof of this
can only be obtained isolating the particles, that is, by obtaining them
separate from everything else (purifying them). In 1984 both Gallo’s and
Montagnier’s groups reported finding polyadenylated (adenine rich) RNA (poly(A)-RNA)
in the 1.16 gm/ml band material obtained from "infected" cultures. The
RNA was claimed to be HIV RNA that is, the HIV genome, and its complementary
DNA, the HIV provirus. However,
as mentioned, although Montagnier claimed his 1.16 gm/ml band contained
pure "HIV"particles, according to him neither his band nor that
of Gallo’s contained any particles with "morphology typical of
retroviruses";
poly(A)-RNA is not specific to retroviruses. It can be found in all
cells and even at the 1.16 gm/ml band obtained from
"non-infected" cells;
there is no proof for the existence of a unique molecular entity,
"HIV-RNA" or "HIV-DNA", while the genomes of the most
variable RNA viruses do not differ by more than 1%. The difference between
the human and the chimpanzee genomes is no more than 2% while there is up
to 40% variation between "HIV" genomes;
in hybridization studies using the "HIV RNA" or cDNA, Gallo
and since then many other researchers have been unable to prove the
existence of the HIV genome in fresh lymphocytes from AIDS patients. In
1994 Gallo stated "We have never found HIV DNA in the tumour cells of
KS…In fact we have never found HIV DNA in T-cells".
All the claims of the existence of HIV in humans are based on polymerase
chain reaction (PCR) studies using small fragments of the "HIV"
genome as primers. However even researchers who believe that there is
proof that the HIV primers and probes used in these studies are HIV accept
that the specificity of this assay, using the antibody as a gold standard,
varies between zero and 100 per cent. Even with the PCR nobody has
reported the existence of the full "HIV" genome in the fresh
lymphocytes of even a single AIDS patient.
Furthermore, despite the ample evidence to the contrary, for most
retrovirologists the finding of a novel nucleic acid in a cell can be due to
nothing else but an infectious agent. Half a century has passed since the Nobel
Laureate Barbara McClintock discovered the phenomenon of transposition which can
lead to the appearance of new genotypes and phenotypes. According to McClintock,
the genome can be restructured not only by transposition but also by other means
as well. In her Nobel lecture of 1983, she said, "rapid reorganisation of
genomes may underline some species formation. Our present knowledge would
suggest that these reorganizations originate from some "shock" that
forced the genome to restructure itself in order to overcome a threat to its
survival...Major genomic restructuring most certainly accompanied formation of
new species". The "genomic shock" which leads to the origin of
new species may be "either produced by accidents occurring within the cell
itself, or imposed from without such as virus infections, species crosses,
poisons of various sorts, or even altered surroundings such as those imposed by
tissue culture. We are aware of some of the mishaps affecting DNA and also
of their repair mechanisms, but many others could be difficult to recognize.
Homeostatic adjustments to various accidents would be required if these
accidents occur frequently. Many such mishaps and their adjustments would not be
detected unless some event or observation directed attention to
them...Unquestionably, we will emerge from this revolutionary period with
modified views of components of cells and how they operate, but only however, to
await the emergence of the next revolutionary phase that again will bring
startling changes in concepts".
As we have mentioned elsewhere9,19 an exogenous retrovirus is only
one possible explanation for the finding of novel nucleic acids in AIDS
patients. Other explanations are:
The genome of an endogenous retrovirus, that is, a stretch of RNA with a
corresponding proviral DNA present in the cellular DNA of uninfected animals
and which is passed from generation to generation vertically (from parents
to offspring via the germ cell line) and which under certain conditions can
be expressed and incorporated into retroviral particles.
The genome of a retrovirus de novo assembled by genetic
recombination and deletion of: (a) endogenous retroviral sequences or (b)
retroviral and cellular sequences or (c) non-retroviral cellular genes.
An RNA obtained by transposition, that is, by certain replicating DNA
sequences (transposons) becoming inserted elsewhere in the genome, or by
retroposition, that is, by particular RNA (retrotransposons) first being
transcribed into DNA and then similarly being inserted into the genome.
Retroposition can "use cellular mechanisms for passive retroposition,
as well as retroelements containing reverse transcriptase". The
retroelements may be retrovirus-like elements or nonviral elements. Not only
can retroposition "shape and reshape the eukaryocytic genome in many
different ways" but the nonviral retroelements may be similar to the
retroviral elements.
A basic principle of molecular biology is that the primary sequence of RNA
faithfully reflects the primary sequence of the DNA from which it is
transcribed. However, in the 1980s RNA editing, "broadly defined as a
process that changes the nucleotide sequences of an RNA molecule from that of
the DNA template encoding it", was discovered. In the process a
non-functional transcript can be re-tailored, producing a translatable mRNA, or
modify an already functioning mRNA so that it generates a protein of altered
amino acid sequences. Sometimes editing is so extensive that the majority of
sequences in a mRNA are not genomically encoded but are generated post-transcriptionally
producing the "paradoxical situation of a transcript that lacks sufficient
complementarity to hybridize to its own gene!". According to Nancy Maizels
and Alan Weiner from the Department of Molecular Biophysics and Biochemistry at
Yale University, "the central dogma has survived hard times. The discovery
of reverse transcriptase amended but did not violate the central dogma of how
genes make proteins; introns qualified the conclusion that genes are necessarily
collinear with the proteins they encode; somatic rearrangement of lymphocyte DNA
called stability of eukaryotic genomes into doubt...and catalytic RNA challenged
the pre-eminence of proteins and breathed new life into the ancient RNA
world". However, the discovery of RNA editing "could come close to
dealing it a mortal blow".
Thus the finding of novel RNAs in human cells, especially those of AIDS
patients and those at risk, can no longer be regarded as incontrovertible proof
that the RNA has been exogenously introduced by a putative HIV or any other
infectious agents. That this may be the case has of late been accepted by Luc
Montagnier. In a written testimony dated February 2nd 2000, to the US
House of Representatives Committee on Government Reform, Subcommittee on
National Security, Veterans Affairs and International Relations, in support of
the work of his colleague, Howard B Urnovitz, (Montagnier is on the scientific
advisory board of a publically traded biomedical company whose director is
Urnovitz), Montagnier wrote: "I have reviewed Dr Urnovitz's published
research and the testimony prepared for presentation to this Committee and
strongly advise that future research on Gulf War Syndrome should include the
study of the detected genetic material".
Urnovitz and his colleagues presented evidence of the existence, in Persian
Gulf War veterans, of "novel", "nonviral" RNAs,
"possibly induced by exposure to environmental genotoxins". They
concluded: "The patterns of the occurrence of RPAs [polyribonucleotides] in
the sera of GWVs [Gulf War Veterans] and healthy controls are sufficiently
distinct to suggest possible future diagnostic applications…Our studies of
patients with active multiple myeloma suggest that patients with individual
chronic multifactorial diseases may have unique RPAs in their sera. Validated
tests for such putative surrogate markers may aid in the diagnosis of such
diseases or in the evaluation of responses to therapeutic modalities".
In the same year that Montagnier claimed HIV isolation, 1983, in a paper
entitled "Expression of novel transposon-containing mRNAs in human T
cells" researchers from the USA and Canada reported "Using an HERV-H
LTR probe, 6 and 4.5 kb transcripts were detected by Northern blot analysis
which were induced in normal peripheral T cells after treatment with
phytohaemagglutin" (HERVº human endogenous
retrovirus). Phytohaemagglutin has been and continues to be used not only by
Montagnier but by virtually every retrovirologist who claims proof for HIV
isolation.
Since Montagnier agrees with Urnovitz that novel, nonviral RNAs appear in the
Gulf War Veterans, then why should the existence of novel RNAs:
In AIDS patients and those at risk be the genome of a retrovirus HIV and
not the result of the many toxins including genotoxins to which they are
exposed?
In cultures containing tissues from AIDS patients be interpreted as HIV
RNAs rather than the result of the many toxins including genotoxins to which
both the patients and the cultures are exposed? Especially when both
Montagnier and Gallo accept that HIV cannot be detected in cultures which
are not treated with such toxins (oxidant agents) including PHA?
When hard pressed all the HIV experts will ultimately accept the
non-specificity of retroviral-like particles, reverse transcription and
antibody/antigen reactions. That this is the case is illustrated is exemplified
by the fact that at the beginning of the AIDS era they were the first to report
isolation of HIV from individuals at no risk of AIDS.166-168 In 1985
Weiss and his colleagues reported the isolation of a retrovirus from
mitogenically stimulated T-cell cultures of two patients with common variable
hypogammaglobulinaemia. This retrovirus "was clearly related to HTLV-III/LAV"
(HIV); evidence included positive western blot with AIDS sera and hybridization
with HIV probes.168 In 1984 Montagnier and his colleagues reported
that "activated lymphocytes from a healthy blood donor were spontaneously
releasing a virus similar to LAV1 in culture."169 With the
method presently used for "HIV" isolation (reaction of p24 with
proteins in culture) Schupbach and his associates using cultures of whole blood
reported positive results in 49/60 (82%) of "presumably uninfected but
serologically indeterminate" individuals and in 5/5 (100%) "seronegative
blood donors".170
CONCLUSION
In 1983 Luc Montagnier and his colleagues and in 1984 Robert Gallo and his
colleagues claimed to have proven the existence of HIV by purifying retroviral
particles, that is, by obtaining a mass of particles isolated from everything
else and showing that the particles are infectious. A critical analysis of their
evidence shows that neither group presented proof of isolation of a novel
retrovirus from AIDS patients. The phenomena they interpreted as HIV are all
non-specific and were known to be so long before the AIDS era. In fact, given
the origin of the cells and the culture conditions one would be expect to find
all these phenomena even if the cultures are not infected with a retrovirus. In
1997 Montagnier himself stressed that to prove the existence of a unique
retrovirus purification is absolutely necessary and admitted that he had not
presented such proof, and in his view, neither had Gallo. Recognition of these
facts may prove the first step in solving the problem of AIDS.
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ATZ and Nevirapine. Perth: The Perth Group,2001. http://aidsmyth.addr.com/report/news/newperthpaper.htm
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HIV and AIDS. Research in Immunology 1992;143:145-8.
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Causer D, Page B. A critical analysis of the HIV-T4-cell-AIDS hypothesis. Genetica
1995;95:5-24.
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Africa: Distinguishing fact and fiction. World Journal of Microbiology and
Biotechnology 1995;11:135-143.
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VIII, HIV and AIDS in haemophiliacs: an analysis of their relationship. Genetica
1995;95:25-50.
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Isolation of HIV: Has it really been achieved? Continuum 1996;4:1s-24s.
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1996;4:24-27.
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transmission by donor semen. Lancet 1996;347:190-1.
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antibodies: Further questions and a plea for clarification. Current Medical
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H, Miller T. A critical analysis of the pharmacology of AZT and its use in AIDS. Current Medical Research and Opinion 1999;15 (Supplement 1):1s-45s.
15. Papadopulos-Eleopulos E, Turner VF. Analysis of mother-to-child
transmission of HIV and the HIVNET 012 (Nevirapine) trial from Uganda, 2001 www.theperthgroup.com/aids
16. Papadopulos-Eleopulos E, Turner VF, Papadimitriou JM, Alfonso H, Page BA,
Causer D, et al. Global voices on HIV/AIDS. Heterosexual transmission of HIV in
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2002;324:1035 /cgi/content/full/324/7344/1034#resp3
17. Papadopulos-Eleopulos E, Turner VF, Papadimitriou JM, Alfonso H, Page BAP,
Causer D, et al. High rates of HIV seropositivity in Africa-alternative
explanation. in press International Journal of STD and AIDS 2003.
Competing interests: None declared
Competing interests:
25 April 2003
Eleni Papadopulos-Eleopulos
Biophysicist
Valendar F. Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso
Department of Medical Physics,Royal Perth Hospital, Western Australia 6001
Rapid Response:
A critical examination of the evidence for the existence of HIV
A critical examination of the evidence
for the existence of HIV
In his rapid response "For some people it’s April Fool’s day all
year round" (3
April 2003) with regards to the Padian
study, Peter Flegg wrote "I explicitly stated that "there was a low
transmission rate" in her study…". It must be pointed out that in
the prospective study of 175 HIV–discordant couples, tested every six months,
not one of the 175 non-infected men or women became HIV positive. That is, the
prospective Padian study did not prove a low transmission rate but a zero
transmission rate, that is, no transmission.
As far as Peter Flegg being perplexed about the thinking of
"dissidents", does he expect all scientists to think alike? Science
develops by a diversity of views and by questioning existing paradigms. While we
agree with the main views held by other "dissidents", that is, there
is no proof that HIV cause AIDS, there are also differences. We also agree with
some views held by the "HIV" experts including the following:
indicates a propensity for the development of some diseases. However,
nowhere in the scientific literature is there evidence that a positive test
proves HIV infection. In other words, the antibody test is similar to the
erythrocyte sedimentation rate (ESR), a laboratory test of considerable
clinical utility that predicts the onset or presence of illness and whose
level is used to monitor the natural history or treatment of diseases.
Nonetheless, it is devoid of specificity.
sex is necessary for the prevention of AIDS. In fact we go one step further.
The HIV experts advise safe sex need be practised only with a positive
partner. We say safe sex should be practised with all partners irrespective
of antibody status.
Peter Flegg also wrote we claim that HIV "spreads through anal
intercourse". In fact, in one of our rapid responses we wrote "It is
also our view that a positive antibody test ("HIV") is acquired by the
same means, that is, passive anal intercourse."( 24th
March 2003) In another rapid response we
wrote "all the presently available data prove it is only passive anal
intercourse which is the risk factor for the acquisition of a positive antibody
test." (1st
April 2003.) This means that either HIV
is the only unidirectionally spread STD in the history of medicine or whatever
causes the positive antibody test cannot be an infectious agent.
It is true that in some of our articles, to present our argument we have
assumed the existence of HIV in order to show how such an assumption leads to
untenable claims for the HIV theory of AIDS.
Peter Flegg wrote that we have been "asserting that HIV does not
exist". Peter Flegg may have interpreted our published material in this
manner but it is important to state that we have never said HIV does not exist.
Rather, from the very beginning when Montagnier and Gallo published their
studies in 1983/1984, we have consistently argued that neither their data nor
anybody else’s prove that HIV exists. (This issue can be resolved by
performing experiments. This is exactly what we argued for and what was accepted
by both sides at the Presidential AIDS Panel Meetings held in July 2000,
Johannesburg, South Africa http://aidsmyth.addr.com/panelreport.htm).
Some of the reasons can be found in what follows.
INTRODUCTION
Following the appearance of AIDS in 1981 many aetiological factors were
proposed. In May 1983 Montagnier announced the discovery of a retrovirus, now
known as HIV, from lymphatic tissue of a gay man with lymphadenopathy. One year
later Gallo reported data which "suggests that HTLV-III [HIV] is the
primary cause of AIDS". By 1986 the scientific community accepted the Gallo
assertion that the same data was "clearcut evidence" that HIV is the
causative agent of the clinical syndrome. Even today the five Science
papers published by these French and American groups are still widely regarded
as proving beyond all reasonable doubt that HIV exists and is the cause of AIDS.
However, not all scientists accepted these findings. In 1987 Peter Duesberg
published an invited paper in Cancer Research on retroviruses and cancer
in which he also questioned the role of HIV in AIDS. At the same time one of us
(EPE) also challenged the theory including the data claimed to prove the
existence of HIV. Papadopulos-Eleopulos also proposed an alternative,
non-infectious aetiology and treatments based on this hypothesis. Since then our
group has published papers addressing every facet of the HIV theory including a
detailed examination of HIV isolation and the HIV genome. Here we confine
ourselves largely to addressing the data published by Montagnier and Gallo in
their 1983/84 Science papers. Genomic data are only briefly discussed
because the existence of HIV and the HIV theory of AIDS were universally
accepted before such data were available. For a comprehensive discussion on
genomic data the reader is referred to reference 19.
RETROVIRUSES AND THEIR IDENTIFICATION
A virus possesses two characteristic properties. The first is anatomical,
that is, being a microscopic particle of individual morphology, the second, the
ability to generate identical progeny by synthetic processes obligatorily
occurring within living cells. It is the latter attribute which defines a
particle with the appearances of a virus, that is, a viral-like particle, as
infectious and thus a virus. The three subfamilies (0ncovirinae, Lentivirinae
and Spumavirinae) of Retroviridae (Retroviruses) are
"enveloped viruses with a diameter of 100-120 nm budding at cellular
membranes. Cell released virions contain condensed inner bodies (cores) and are
studded with projections (spikes, knobs)". The retroviral particles contain
RNA and the enzyme reverse transcriptase (RT), an RNA dependent DNA polymerase
which catalyses the synthesis of DNA contrary to the central dogma of biology,
that is, in a direction "reverse" from DNA to RNA. According to
retrovirologists, such DNA is then integrated into existing cellular DNA as a
"provirus". Retroviral particles share the property of concentrating
(banding) at a density of 1.16 gm/ml when centrifuged at high speeds in sucrose
density gradients, a fact long used in their purification.
All retrovirologists agree that to prove the existence of a new retrovirus
one must isolate it. However, the term "virus isolation" is beset with
semantic difficulties and ambiguities. The dictionary meaning of
"isolation" derives from the Latin insulatus (made into an
island) and refers to the act of separating an object from all other matter that
is not that object. "Purification" means to obtain something free from
impurities. In this context isolation is the same as purification. Because virus
particles are small it is not possible to obtain a single, isolated particle.
The next best thing is to obtain a mass of particles separate from everything
else. Until the early 1980s, for the isolation of animal retroviruses as well as
the "first" human retrovirus HL23V, by isolation retrovirologists
meant purification. On the other hand, nowadays both basic and specialised texts
rarely define "isolation" and when they do such attempts are
non-illuminating. For example, Levy defines isolation as a "sample of a
virus from a defined source", and White as the ability to "identify a
totally unforeseen virus, or even discover an entirely new agent".
Encompassed as "virus isolation" are listed methods of culturing
specimens in tissue and chick embryo cells, as well as live animals, following
by documentation therein of cytopathic and pathological effects, haemoabsorption,
immunofluorescence, antigen/antibody reactions and "characterisation of the
viral genome". HIV experts, including Luc Montagnier and Robin Weiss define
"virus isolation" as "propagating them [viruses] in cells in
culture" and see www.theperthgroup.com/aids/vftweiss.html.
However, if "virus isolation" is to "take a sample of a virus
from a defined source", or "propagating them in cells in
culture", then first one must have prior proof that a virus exists in
"a defined source" or "in cells in culture". One cannot know
that a virus exists or define its constituents without purification (isolation)
of the putative viral particles.
There are several reasons why this is mandatory:
To prove that the retrovirus-like particles are infectious, that is, they are
a virus
The finding of particles with the appearances of retroviruses, is not proof
that such particles are retroviruses and even less proof a particle is a
particular retrovirus. Particles bearing the morphological characteristics of
retroviruses are ubiquitous. In the 1970s such particles were frequently
observed in human leukaemia tissues, cultures of embryonic tissues and "in
the majority if not all, human placentas". Type-C retroviral particles are
present in "fish, snakes, worms, pheasant, quail, partridge, turkey,
tree-mouse and agouti" as well as in "tapeworms, insects...and
mammals". Gallo was well aware of this problem as far back as 1976 when he
wrote: "Release of virus-like particles morphologically and biochemically
resembling type-C virus but apparently lacking the ability to replicate have
been frequently observed from leukaemic tissue". In other words, it is not
possible to claim a particle is a retrovirus merely by appearances. To prove
that retrovirus-like particles observed in a culture are a virus one must
isolate the particles, characterise their proteins and RNA and introduce the
particles into a secondary culture. If any particles are released in the
secondary culture they too must be isolated and proven that their proteins and
RNA are the same as those from the primary culture. In such experiments one must
not ignore the use of legitimate controls and in doing so take in consideration
an important difference between retroviral and other infectious agents.
When one finds an infectious agent, for example a virus or a bacterium,
either in vitro or in vivo, one may be assured that the agent has
been introduced into the culture or animal from outside. Retroviruses are the
exception. This is because normal human and animal genomes contain information
which, under the appropriate conditions, leads to the synthesis of retroviral
RNA and proteins, or even to the assembly of retroviral particles, that is, to
the expression of endogenous retroviruses. And although as late as 1994 both
Gallo and Fauci taught "there are no known human endogenous
retroviruses", it is known that at least 1% of the human DNA is retroviral
DNA and that endogenous retroviruses are present "in all of us".
Furthermore, new endogenous retroviral genomes may arise from rearrangements of
existing retroviral genomes, cellular DNA or both, caused by many factors,
including pathogenic processes. The expression of endogenous retroviral genomes
may arise spontaneously and may be significantly accelerated and the yield
increased by conditions which induce cellular activation. According to the
eminent retrovirologist George Todaro, "the failure to isolate endogenous
viruses from certain species may reflect the limitations of in-vitro
cocultivation techniques". Endogenously produced retroviruses are
morphologically and biochemically indistinguishable from exogenous retroviruses.
Because of this, the finding of identical retrovirus in serially
"infected" cultures/cocultures is not proof that the cells are
infected with exogenous retrovirus. One method which may assist resolve but will
not prove whether cells acquire virus from the outside (exogenously acquired
retrovirus, infectious retrovirus) and have not assembled a retrovirus from
information already existing in normal cells (endogenous retrovirus), is to
conduct control cultures/cocultures in parallel with test cultures/cocultures.
The only difference between test and control cultures should be the introduction
of tissue assumed infected into the test cultures. In every other respect
control cultures must be dealt with identically. For example:
retroviral particles depends on the metabolic state of the cells, the
physiological state of the cells used in the control cultures should be as
close as possible to the test culture;
retroviral particles, if test cells are cocultured, so should the controls;
may increase endogenous retroviral expression. Because of this, when host
cells are cultured with supernatant or material which bands at 1.16 gm/ml
from cultures thought to be infected, the controls must be cultured with
similar material from noninfected cultures;
yield increased a million fold by stimulating the cultures with mitogens,
mutagens, chemical carcinogens and radiation, if test cultures are exposed
to or employ such agents so should the controls;
examination of test and control cultures/cocultures should be performed.
To determine their biological effects
Without recourse to pure particles it is impossible to determine whether
effects are due to virus particles or contaminants including "chemical
stimulants", a caveat stressed as far back as 1911 by the Peyton Rous, the
father of retrovirology.
In 1911 Rous induced malignancy in chickens by injections of cell-free
filtrates obtained from a muscle tumour. Similar experiments were repeated by
many researchers and the tumour inducing filtrates became known as filterable
agents, filterable viruses, Rous agents, Rous virus and ultimately retroviruses.
However, Rous himself expressed doubts that the agents which caused the tumours
were infectious in nature. Indeed he warned, "The first tendency will be to
regard the self-perpetuating agent active in this sarcoma of the fowl as a
minute parasitic organism. Analogy with several infectious diseases of man and
the lower animals, caused by ultramicroscopic organisms, gives support to this
view of the findings, and at present work is being directed to its experimental
verification. But an agency of another sort is not out of the question. It is
conceivable that a chemical stimulant, elaborated by the neoplastic cells, might
cause the tumour in another host and bring about in consequence a further
production of the same stimulant".44
To characterise the viral proteins
The only way to prove that a protein is a constituent of an object is to
obtain it from that object, or when the object is very small as is the case of
viruses, from material consisting of purified virus particles. If the material
contains impurities which are proteins or contain proteins, it is not possible
to determine which are viral and which are not. Yet only after the viral
proteins are characterised is it possible to employ them as antigens in antibody
tests.
To characterise the viral genome
As for viral proteins the only way to prove that a stretch of RNA is viral it
is to obtain it from material which contains nothing else but virus particles.
If the material contains impurities the impurities must not include RNA. Then
and only then can the RNA and its complementary DNA (cDNA) be used as probes and
primers for genomic hybridisation and PCR studies.
To act as a gold standard for the antibody tests
The reaction of a virus or viral protein with an antibody present in a
patient's serum does not prove that the antibody is induced by or directed
against the virus or a viral protein. That is, it does not prove the reaction is
specific. This is because there are significant obstacles which hinder the
interpretation of antibody/antigen reactivity including non-specific
stimulation, cross-reactivity or both. Cross reactivity results from antibody
molecules, even monoclonal antibodies, interacting not only with the inducing
antigen but also with other antigens. Indeed, there are instances where
"cross-reactive antibodies may have higher affinity with antigens other
than the inducing antigen. Even antigens that differ for most of their structure
can share one determinant, and a monoclonal antibody recognising this site would
then give a 100% cross-reaction. An example is the reaction of autoantibodies in
lupus with both DNA and cardiolipin...It should be emphasised that sharing a
"determinant" does not mean that the antigens contain identical
chemical structures, but rather that they bear a chemical resemblance that may
not be well understood, for example, a distribution of surface charges".
Since polyclonal antibodies are composites of monoclonal antibodies these facts
apply equally, if not more so, to polyclonal antibodies. These facts have been
extensively exploited in clinical medicine for the diagnosis of diseases such as
syphilis and infectious mononucleosis. In these diseases, T. pallidum and
Epstein-Barr virus cause the appearance of antibodies reactive with ox-heart
proteins and sheep and horse red blood cells. However, this does not mean that
patients are "infected" with ox-heart, or horse red blood cells and
the diseases are induced by these agents. The only way to determine the
specificity of an antibody/antigen reaction is to use an independent method, a
gold standard to prove the presence or absence of the antigen. The only possible
gold standard for a test to prove a virus infection is the virus in question.
That is, virus isolation/purification.
METHODS FOR THE ISOLATION/PURIFICATION OF RETROVIRUS-LIKE PARTICLES
Up till the 1950s retroviruses were isolated/purified by filtration although
this method was less than satisfactory. With the development of the electron
microscope, apparently, for some retrovirologists, the detection of
retrovirus-like particles was deemed sufficient to prove the existence of a
retrovirus. However, other scientists including the well-known retrovirologist,
JW Beard, recognised that cells, including uninfected cells, under various
conditions, were responsible for the generation of a heterogenous array of
particles some with the appearances of retroviruses. Beard stressed:
"identification, characterisation, and analysis are subject to well-known
disciplines established by intensive investigations, and the possibilities have
by no means been exhausted. Strangely enough, it is in this field that the most
frequent shortcomings are seen. These are related at times to evasion of
disciplines or to their application to unsuitable materials. As was foreseen,
much of the interest in the more tedious aspects of particle isolation and
analysis has been diverted by the simpler and undoubtedly informative processes
of electron microscopy. While much can be learned quickly with the instrument, it
is nevertheless clear that the results obtained with it can never replace, and
all too often may obscure, the need for the critical fundamental analyses that
are dependent on access to homogenous materials" (italics ours).
By the 1970s there was general agreement that "Virions of RTV
[retroviruses] have a characteristic buoyant density, and centrifugation to
equilibrium in density gradients is the preferred technique for purification of
RTV". The method of banding in density gradients is not ideal either.
Substances other than retroviruses may band at the same density. This is why at
a meeting held at the Pasteur Institute in 1972, Francoise Barre-Sinoussi and
Jean-Claude Chermann stressed that to claim purification of retrovirus-like
particles using sucrose density gradients it is absolutely necessary to prove,
using the electron microscope, that the 1.16 gm/ml band contains nothing else
but particles with "no apparent differences in physical appearances".
THE PHENOMENA CLAIMED TO PROVE THE EXISTENCE OF HIV
In May 1983 Luc Montagnier, Francoise Barre-Sinoussi, Jean-Claude Chermann
and colleagues published a paper in Science entitled, "Isolation of
a T-Lymphotropic retrovirus from a patient at risk for Acquired Immune
Deficiency Syndrome (AIDS)". This is the paper which, since the resolution
of the polemics between Montagnier and Gallo concerning allegations of
misappropriation by the latter of the French virus sent to the US by the Pasteur
Institute, is accepted as being the study which proved the existence of HIV.
There it was shown that mitogen stimulated lymph node cell cultures from a gay
man (BRU) with lymphadenopathy were able to transcribe the synthetic RNA
primer-template An.dT15. From this data Montagnier and his colleagues
concluded that BRU’s lymph node cells were infected with a retrovirus. The
finding of the same activity in the supernatant of a coculture consisting of the
same cells with stimulated lymphocytes from a healthy individual was considered
proof for virus transmission as well as isolation. In another experiment
supernatants from the cocultures were added to two, three day old, stimulated
umbilical cord lymphocytes cultures. "Electron microscopy of the infected
umbilical cord lymphocytes showed characteristic immature particles with dense
crescent (C-type) budding at the plasma membrane". Supernatant from the
culture was banded in sucrose density gradient and the 1.16 gm/ml band was shown
to transcribe An.dT15. The proteins in the 1.16 gm/ml band as well as
the proteins of a cellular extract were separated according to their molecular
weight using "denaturing buffer and electrophoresed on 12.5 percent
polyacrylamide-SDS slab gel". When the strips were incubated with human
sera many proteins from the cellular extracts were found to react with serum
from BRU, another gay man as well as a "healthy donor". In the strips
containing the proteins from the 1.16 gm/ml band three proteins including a p25
(‘p’ for protein, 25 for its molecular weight in thousands) were found to
react. They also reported that the p25 did not react with antibodies to HTLV-I.
The material banding at 1.16 gm/ml was claimed to be "purified, labelled,
virus" although no electron microscopic data were presented. The authors
concluded: "A retrovirus belonging to the family of recently discovered
human T-cell leukaemia viruses (HTLV), but clearly distinct from each previous
isolate, has been isolated from a Caucasian patient with signs and symptoms that
often precede the acquired immune deficiency syndrome (AIDS). This virus is a
typical type-C RNA tumor virus, buds from the cell membrane, prefers magnesium
for reverse transcriptase activity, and has an internal antigen (p25) similar to
HTLV p24".
Robert Gallo and his associates did not consider the Montagnier group data as
proving "true isolation". As late as 1997, in a book published by one
of the best known HIV experts, Jaap Goudsmit, one reads: "The BRU lymph
node was first cultured in early January 1983 and, on January 15, it shed an
enzyme absolutely unique to the lentivirus group. [The enzyme is not even
specific to retroviruses much less to Lentiviruses (see below)]...The BRU
virus grew slowly and with difficulty, but its identity and activity were
reported in the May 20, 1983 issue of Science...The Pasteur Group was
widely acclaimed but very worried. In the world of virology, finding a new virus
is not enough: You must propagate and isolate the organism for analysis by other
virologists. The French had not yet isolated their new lentivirus".
Why then did: (a) Gallo, who reviewed the Montagnier manuscript, recommend
its publication? (b) all the HIV experts including Gallo and Goudsmit (on page
24 of his book one reads: "BRU was the first strain to be isolated")
accept that the first isolation of HIV and thus of its existence was proven in
the May 1983 Science paper?
A year later, in May 1984, Gallo, Popovic and their colleagues published four
papers in Science in which they claimed "isolation" of another
retrovirus from AIDS patients. However, in addition to the use of a leukaemic
cell line, the only difference between the Montagnier and Gallo groups’ data
were quantitative. In the first paper entitled "Detection, Isolation and
Continuous Production of Cytopathic Retroviruses (HTLV-III) from Patients with
AIDS and pre-AIDS", (HTLV-III=HIV), experiments were described in which
"concentrated culture fluids harvested from short-term [mitogenically
stimulated] cultures of T-cells" from patients with AIDS or pre-AIDS were
cultured with a mitogenically stimulated leukaemic cell line HT and highly
selected clones obtained by culturing HT with irradiated cells of a healthy
donor. The data presented as proof of isolation of HIV were: (a) RT activity in
cell free supernatants and the 1.16 gm/ml band; (b) reaction in the cultures
with "Rabbit antiserum to HTLV-III" and "Patient serum (E.T.)";
(c) EM showing the presence of retroviral-like particles in the cultures.
An enquiry conducted by the National Institutes of Health Office of
Scientific Integrity found that the HT cell line was cultured not with
concentrated fluids (supernatant) originating from individual AIDS patients, but
with concentrated fluids pooled initially from individual cultures of three
patients and ultimately from the individual cultures of ten patients. In
evidence given to this enquiry the reason given was because none of the
supernatants "individually was producing high concentrations of reverse
transcriptase". In other words, Gallo and his colleagues did not regard the
levels of RT from individual cultures as proof that individual specimens
contained a retrovirus. The Gallo investigation found the pooling of specimens
to be "of dubious scientific rigor". One scientist described the
procedure as "really crazy". Most importantly how could Gallo use the
reaction with rabbit antiserum to prove HIV isolation (purification) when, to
obtain rabbit antiserum rabbits must first be injected with pure HIV? It is
inexplicable how Gallo and his colleagues could already possess "Rabbit
serum to HTLV-III" before they had proved the existence of a novel virus.
However, if the antiserum was manufactured "from rabbits infected
repeatedly with disrupted HTLV-III", that is, the material banding at 1.16
gm/ml, one would expect to obtain antibodies to all the proteins constituting
this material even if the proteins were cellular and not viral.
In the second paper entitled "Frequent Detection and Isolation of
Cytopathic Retroviruses (HTLV-III) from patients with AIDS and at risk of
AIDS", Gallo and his colleagues claimed to have "isolated" HTLV-III
(HIV) from 26/72 (36%) of AIDS patients. In this paper isolation was defined as
"more than one of the following": "repeated detection of a Mg2+-dependent
reverse transcriptase activity in supernatant fluids; virus observed by electron
microscopy (EM) [retrovirus-like particles in the culture]; intracellular
expression of virus-related antigens detected with antibodies from seropositive
donors or with rabbit antiserum to HTLV-III; or transmission of particles".
Transmission of particles was defined as "detected by RT assays or by
electron microscopic observation, to fresh human [umbilical] cord blood, bone
marrow, or peripheral blood T lymphocytes", cultured with supernatants from
the "infected" cultures. (It can be seen that the Gallo group method
permitted "isolation" of a retrovirus without evidence for either
particles or RT).
In the third paper, proteins from the 1.16 gm/ml band which they claimed was
"purified" HIV, as well as the proteins from the "infected"
cells, were "lysed and fractionated by electrophoresis on a 12%
polyacrylamide slab gel in the presence of SDS. The protein bands were
electrophoretically transferred to a nitrocellulose sheet" and reacted with
different sera. In other words Gallo used a technique which is known as Western
blot (WB) antibody test. Many proteins from the cellular extract were found to
react with sera from both patients and healthy individuals. They also reported
that two proteins from the 1.16 gm/ml band, p24 and p41, reacted with patient
sera. For this and no other reason it was claimed that "these molecules are
the major components of the virus preparation. p24 and p41 may therefore be
considered the viral structural proteins". As far as morphology is
concerned, the Gallo group reported that the HIV particle "is produced in
high numbers from infected cells by budding from the plasma membrane. A possible
unique feature of this virus is the cylindrical shaped core observed in many
mature virions...HTLV-III is a true member of the HTLV family". (HTLVs are
type C retroviral particles and not Lentiviruses as HIV is claimed to
be).
In the fourth paper, instead of separating the proteins which banded at 1.16
gm/ml and then incubating them with patient sera, the mixture of all proteins
was used, that is, they performed a test known as enzyme-linked immunosorbent
assay (ELISA). "To understand the molecular nature of the antigens
recognised by ELISA", they also performed WB, with some of the sera. They
reported that: "Serum samples from 88 percent of patients with AIDS and
from 79 percent of homosexual men with signs and symptoms that frequently
precede AIDS, but from less than 1 percent of heterosexual subjects, have
antibodies reactive against antigens of HTLV-III. The major immune reactivity
appears to be directed against p41, the presumed envelope antigen of the virus…The
data presented here and in the accompanying reports suggest that HTLV-III is the
primary cause of AIDS". Two years later Gallo wrote that "The results
presented in our four papers provided clearcut evidence that the aetiology of
AIDS and ARC was the new lymphotropic retrovirus, HTLV-III"67
(italics ours).
COMMENTS
As mentioned, apart from the quantitative difference, the Gallo group
experiments are no different from those performed by Montagnier and his
colleagues. It follows then, that if Montagnier's group data do not prove
"true isolation" neither did Gallo's nor anybody else’s because to
date everybody has repeated (for the vast majority only part thereof) the same
experiments as these two groups. It is of pivotal significance that neither
group reported the use of valid controls (see above) nor did they prove they had
obtained purified retrovirus-like particles. The question then is, do the data
obtained by the Montagnier and Gallo group, that is RT, particles in culture and
antigen/antibody reactions prove the isolation of a unique, or even a
retrovirus, any retrovirus?
Without doubt, if by isolation one expects proof of purification, then the
detection of an enzyme, or retrovirus-like particles in a culture, or proteins
either in the cells or the 1.16 gm/ml band which react with antibodies present
in human or animal sera, do not comply. To argue otherwise one must define the
detection of cardiac or hepatic enzymes in the blood of patients suffering chest
pain or jaundice as proof for isolation of the human heart or liver. Likewise,
if an antibody/antigen reaction is proof for isolation of a virus then antibody
reactivity to the protein βHCG is proof for isolation of the human
placenta. The assertion that the detection of a retrovirus-like particle in a
culture proves isolation is no different from asserting that the detection of a
fish-like creature in the ocean is the same as having a definite fish in your
frying pan. The detection of RT, retrovirus-like particles and antibody/antigen
reactivity can only be considered proof for the detection of a retrovirus, and
then if, and only if, there is prior knowledge that the three phenomena are
specific to retroviruses.
To claim that the detection of this phenomena proves the existence of a new
retrovirus one must have proof that at least one of the three phenomena is
different from that observed in all other known retroviruses.
Reverse transcriptase
At present some of the leading HIV researchers consider RT as being the
"sine qua non" of retroviruses and regard the detection of
reverse transcription in lymphocyte cultures from AIDS patients not only as
proof of the presence of such viruses but of HIV itself including HIV isolation
and quantification. However, according to some of the best known
retrovirologists including its discoverer, as well as the Nobel Laureate and
former Director of the US National Institute of Health Harold Varmus, reverse
transcriptases are present in all cells as well as bacteria and viruses.
"Reverse transcriptase (RT) was first discovered as an essential catalyst
in the biological cycle of retroviruses. However, in the past years, evidence
has accumulated showing that RTs are involved in a surprisingly large number of
RNA-mediated transcriptional events that include both viral and nonviral genetic
entities". Even if RT were a property only of viruses it is not specific to
retroviruses. According to Varmus, "Reverse transcription was assigned a
central role in the replication of other viruses [hepatitis B and cauliflower
mosaic viruses] and in the transposition and generation of other kinds of
eukaryotic DNA". "The hepatitis B viruses (HBVs) are small DNA viruses
that produce persistent hepatic infections in a variety of animal hosts and
replicate their DNA genomes via reverse transcription of an RNA intermediate.
All members of this family contain an open reading frame (ORF), "P"
(for pol), which is homologous to retroviral pol genes" [pol=polymerase].
"Hepatitis B virus (HBV) resembles retroviruses, including HIV, in several
respects. In particular, both viruses contain reverse transcriptase, and
replicate through an RNA intermediate". Because of this, it has been
suggested that hepatitis B infection should be treated with the same
antiretroviral agents as HIV infection.
At present, evidence exists which shows that although the major target organ
for hepatitis B virus is the liver, cells other than hepatocytes "including
peripheral blood lymphocytes and monocytes, may become infected with HBV".
Lymphocyte stimulation in general and PHA (an agent employed in the majority of
cultures with tissues from AIDS patients), is associated with the production of
hepatitis B virus from peripheral blood lymphocytes in patients infected with
HBV including "viral replication in chronic hepatitis B infection of
childhood". Hepatitis B virus infection is widespread in the AIDS risk
groups.
In the early 1970s Gallo proved that cultures of leukaemic cells transcribe
the An.dT15 template-primer as does material banding at 1.16 gm/ml
originating from "PHA stimulated (but not unstimulated normal human blood
lymphocytes". Reading the Gallo 1984 Science papers the impression
is gained that the leukaemic HT cell line and thus its clones, including H9
which Gallo used, was a new cell line developed in Gallo's laboratory. However
it is now known that the HT cell line is the HUT78 cell line which originated
from a patient with adult T cell leukaemia, a disease which Gallo claims is
caused by the retrovirus, HTLV-I. A year earlier Gallo claimed to have proven
that the HUT78 cells are infected with HTLV-I. If this is the case, the Gallo
group would have detected RT in their cultures even if the enzyme is specific to
retroviruses and the cultures were not infected with HIV. Other researchers
reported RT activity in normal non-infected spermatozoa.
It must also be pointed out that the presence of HIV reverse transcriptase
was detected by both Montagnier and Gallo, (and all HIV experts since)
indirectly, that is, by detecting the transcription of the template-primer An.dT15.
However, at least in 1984 if not 1983, Montagnier and his colleagues knew that
in the 1970s there was proof that "Among a number of template primers, (rA)n.(dT)12-18
has been most frequently employed since RT shows high activity with this
template primer. However, the problem is that the cellular DNA polymerases (pol b
and pol g ) also effectively utilize the same
template primer". In fact, in 1975, an International Conference on
eukaryotic DNA polymerases defined DNA polymerase γ as the cellular enzyme
which "copies An.dT15
with high efficiency but does not copy DNA well". One year earlier Gallo
wrote: "Under appropriate reaction conditions DNA polymerase β can
efficiently transcribe poly (A) primed by oligo (dT)". Thus it is possible
to detect transcription of An.dT15 even when no RT, either viral or
cellular, is present, especially under the conditions used to prove the
existence of HIV. (Both Montagnier and Gallo accept that the phenomena detected
in cultures which are said to prove HIV isolation cannot be detected unless the
cells are chemically stimulated). Nowadays the non-specificity of RT is
broadcast even in the popular press to readers contemplating the purchase of
shares in biotechnology companies.
In conclusion, even if one accepts Montagnier and Gallo's groups’
definition of isolation, given that RTs and reverse transcription are
nonspecific to retroviruses, their detection even in an unlimited number of
consecutive cultures/co-cultures cannot be considered proof for isolation and
propagation of a retrovirus.
"HIV" particles
Montagnier and his colleagues reported HIV initially as a type C particle,
then as a type D particle and then as a Lentivirus. In 1984 Gallo and his
colleagues reported HIV as a type C particle. However, in 1985 Gallo wrote:
"A possible unique feature of the virions is the cylindrical core observed
in many presumably mature virions. Virions having this type of core have been
frequently reported for certain type D retroviruses, and in some instances, for
type C retroviruses". Jay Levy reported HIV as a type D particle. Others at
the University of California wrote that "AIDS virus isolated show
morphologic characteristics of type C, type D and Lentiviruses". According
to Anthony Fauci and others" "T-cells and macrophages handle the virus
very differently. In the T-cell, virus buds out of the external plasma membrane
of the cell. In the monocyte/macrophage cultures it buds into membrane-bound
vesicles inside the cells". The latter is a description of a type A,
retroviral particle. Thus the leading HIV experts have described HIV as a member
of two subfamilies and three genera of Retroviridae. These taxonomical
differences imply that if HIV was a newly discovered mammal, it could have been
either human, a gorilla or an orang-utan. By consensus at present HIV is
regarded as a Lentivirus. This agreement was reached when it was realised
that in "HIV" positive individuals AIDS did not appear soon after
"infection", although the designation Lentivirus is a
morphological description.
"HIV infected cultures" contain in addition to the particles with
the morphology attributed to HIV many other "viral particles". For
example: Hockley and his colleagues from the Electron Microscopy and Photography
Section and Division of Virology at the National Institute for Biological
Standards and Control in the United Kingdom describe a profusion of particles
which they divide broadly into three groups, mature, ring-like and small with
spikes. The mature particles "were approximately spherical in shape and 100
to 150nm in diameter. The outer lipid membrane was frequently broken or absent
in places, and there was no evidence of surface spikes...A few mature particles
were found that were larger than average and appeared to contain a double
nucleoid...in the preparation of HIV there were always many vesicles with
granular contents in which it was not possible to recognise a distinct nucleoid".
Also, "The ring-like particles had a more consistently spherical shape and
were larger (140nm in diameter)" and the small particles "were
unusually spherical but sometimes slightly angular in shape and 65 to 90nm in
diameter" and had spike-like projections on their surface. Hans Gelderblom
who has done most of the EM studies in HIV/AIDS research reported that although Lentiviruses
and thus HIV is considered to have a cone shaped core, he and his colleagues
found centrosymmetric and tubular cores as well. The caption to one photograph
reads: "Virions can be seen having either elongated, 'baton-like' tubular
cores 30-35nm in diameter or containing more than one core. Tubular and regular
cone-shaped cores can coexist within one virion". The text states:
"Rarely, tubular core structures reminiscent of batons with a diameter of
30-35nm and a length of 150-250nm are observed". Lekatsas and other
virologists from Pretoria and Johannesburg reported: "We used the
characteristic cylindrical structure in the core as an identifying
characteristic for the virus to distinguish it from cellular debris and also
noted that it may vary considerably in its dimensions and morphological
features. We have found two basic virus particle sizes, 90nm and 120nm, both
present in large numbers. The larger particle bears no surface projections while
the smaller particle is rarely 'naked' and usually bears projections". The
US CDC reported: HIV particles are "usually round and have a diameter of
about 85-95nm...Virus with bar-shaped nucleoids and particles with a tear-drop
shape are commonly seen in HTLV-III/LAV infected lymphocytes, sometimes
ring-shaped particles without dense nucleoids are also seen". The question
then arises if the particles with the "unique" morphology considered
to be HIV represent an exogenous retrovirus originating from tissues of AIDS
patients or those at risk, then what is the origin and role of the many non-HIV
particles and which, if any, of the "HIV" or non-HIV particles band at
1.16 gm/ml? That is, which have the density characteristic of retroviruses?
Retrovirus-like particles have been found in non-HIV-infected cord blood
lymphocytes cell cultures and in cells used for HIV "isolation" such
as and H9 (HUT-78), CEM, C8166 and EBV transformed B-cells. Retrovirus-like
particles antigenically related to HIV have been found in cultures of salivary
gland extracts from patients with Sjorgen's syndrome. In the only EM study,
either in vivo or in vitro in which suitable controls were used
and in which extensive blind examination of controls and test material was
performed, particles indistinguishable from "HIV" were found in 18/20
(90%) of AIDS as well as in 13/15 (87%) of non-AIDS related lymph node
enlargements. This led the authors to conclude: "The presence of such
particles do not, by themselves indicate infection with HIV".
It is of pivotal significance to note that:
has reported the presence of "infectious plasma HIV".
both principle morphological characteristics of retroviruses, that is,
"a diameter of 100-120nm" AND surfaces which "are studded
with projections (spikes, knobs)". The electron microscopist agree that
the "HIV" particles are devoid of knobs and thus of the
"HIV" protein gp120 (see below) said to be the constituent protein
of the knobs.
Hans Gelderblom and his colleagues have estimated that immediately after
being released from the cell membrane "HIV particles" possess an
average of 0.5 knobs per particle which are rapidly lost, but also pointed out
that "it was possible that structures resembling knobs might be observed
even when there was no gp120 [knobs] present, i.e. false positives".Yet
all HIV experts agree that the infectivity of the HIV particles is determined by
the gp120 (knobs). Thus, according to Montagnier et al, "The gp120
is responsible for binding the CD4 receptor" while for Matthews and
Bolognesi, "First gp120 binds to the CD4 receptor on an uninfected cell;
then gp41 becomes anchored in the adjoining membrane; next the two membranes
begin to fuse, and the virus spills its contents into the cell". Callebaut et
al state, "The human immunodeficiency virus (HIV) infects lymphocytes,
monocytes, and macrophages by binding to its principal receptor, the CD4
molecule, through the viral envelope glycoprotein gp120. The V3 loop of gp120 is
critical for HIV infection". Others are in complete agreement.
The general agreement that gp120 (knobs) is absolutely necessary for HIV
particles to be infectious and the fact that this protein (knobs) is not present
in the cell free particles leads to one unavoidable conclusion, that is, the
"HIV" particles are not infectious, they are not viral particles.
Furthermore,
Gallo used cell-free fluids (supernatants). Montagnier cultured umbilical
cord lymphocytes with supernatant from the cocultures of lymphocytes from
BRU cultured with lymphocytes from a healthy individual. Gallo cultured the
HUT-78 cell line with supernatants from the cultures of ten
"infected" individuals. Even if the supernatants contained
particles, being cell free the particles would be devoid of knobs (gp120),
that is, they would not have been infectious. This means that even if
Montagnier and Gallo had proof that their umbilical and HUT-78 cloned
cultures contained particles with all the morphological characteristics of
retroviruses, that in sucrose density gradients the particles banded at the
1.16 gm/ml band and the band contained nothing else but particles, the
particles could not have originated from their patients. Furthermore, since
the only samples which the Pasteur Institute gave to the Gallo laboratory
were cell free, one must question the basis upon which Gallo was accused of
misappropriation of the French virus.
contaminated factor VIII. However, to date nobody has reported the presence
of "HIV" particles in plasma. Even if such particles were present
the particles would be devoid of gp120. Since gp120 is "crucial to
HIV's ability to infect new cells" it is not possible for haemophiliacs
to be infected with HIV through factor VIII administration. There is another
fundamental reason why it is impossible for haemophiliacs to be infected
with "HIV" from "contaminated" factor VIII. According to
a publication from the CDC,112 "In order to obtain data on
the survival of HIV, laboratory studies have required the use of
artificially high concentrations of laboratory grown virus...the amount of
virus studied is not found in human specimens or any place else in
nature,...it does not spread or maintain infectiousness outside its host.
Although these unnatural concentrations of HIV can be kept alive under
precisely controlled and limited laboratory conditions, CDC studies have
shown that drying of even these high concentrations of HIV reduces the
number of infectious viruses by 90 to 99 percent within several hours. Since
the HIV concentrations used in laboratory studies are much higher than those
actually found in blood or other body specimens, drying of HIV-infected
human blood or other body fluids reduces the theoretical risk of
environmental transmission to that which has been observed--essentially
zero". Given that factor VIII is dispensed as a freeze-dried powder
which spends many weeks or months waiting use, it is incomprehensible that
the CDC and others continue to regard patients with haemophilia at risk for
HIV infection via contaminated factor VIII concentrates and enigmatic that
another explanation for "HIV" and AIDS in haemophiliacs has not
been sought.4
The only conclusion one can draw from the electron microscopy data is that
the reported particles are non-HIV or even retroviral specific which means that
the detection of such particles, be it in an unlimited number of consecutive
cultures/cocultures, is not proof for isolation, no matter how isolation is
defined.
Furthermore:
specific to retroviruses, they are not specific for a unique retrovirus. The
only evidence both groups presented for the existence of a unique
retrovirus, HIV, is the antigen/antibody reaction, as acknowledged by both
Montagnier and Gallo.
under the conditions used by Montagnier and Gallo is not proof that the
retrovirus is present in vivo. The only evidence which both Gallo and
Montagnier's groups presented for the existence of HIV in vivo was
the antigen/antibody reaction.
is a "correlation" between the antibody test and the appearance of
AIDS.
is crucial for the HIV hypothesis of AIDS and in fact for the existence of a
unique retrovirus, HIV;
question not only the HIV hypothesis of AIDS but also the existence of HIV.
"HIV" antibodies
It has already been mentioned that the mere interaction between an antigen
such as a protein or a virus and an antibody does not prove any more than a
chemical relationship. In all other aspects the reaction may be totally
nonspecific. To prove the specificity of the reaction one must employ an
alternative, independent method of proving the existence of the antigen, that
is, one must use what is generally known as a gold standard. The only possible
gold standard for the HIV antibody tests (the antigen/antibody reaction) is HIV
itself, that is, HIV isolation. No matter how one defines isolation, obviously
the definition cannot include the antibody/antigen reaction (the antibody test,
neither the WB nor the ELISA) because, by doing so, not only does one not have
an independent analysis, the test becomes its own gold standard and is thus
rendered meaningless. Reading the 1984 Gallo Science papers it appears
that Gallo always defined isolation as "more than one of the following:
reverse transcriptase activity either in the supernatant or the 1.16 gm/ml band;
retrovirus-like particles in the culture or reaction between proteins (either in
the cultured cells or the 1.16 gm/ml band) with antibodies present in the
patients sera or "antiserum to HTLV-III". However, in an interview
Gallo gave to Huw Christie, the editor of the British magazine Continuum,
at the 1998 Geneva AIDS Conference, Gallo said, "Sometimes we had Western
Blot positive but we couldn't isolate the virus. So we got worried and felt we
were getting false positives sometimes so we added the Western Blot. That's all
I can tell you. It was an experimental tool when we added it and for us it
worked well because we could isolate the virus when we did it". In other
words:
must use a gold standard.
isolation, the definition did not include the antibody/antigen reaction (the
Western Blot). This means that Gallo and his colleagues were aware that an
antibody/antigen reaction cannot be used as proof for isolation. Yet, by the
time their Science papers were published, and in order to reconcile
the low correlation between what they initially called isolation and the
antibody/antigen reaction, arbitrarily, and against all scientific
reasoning, they "added the Western Blot" to their definition of
isolation. (It is interesting that even with their novel definition the
correlation between "isolation" and antibody tests was still less
than perfect. They "isolated" HIV from 26/72 (36%) of patients
with AIDS, while 88% of patients with AIDS were seropositive using an
antibody test Gallo considered highly specific).
In an interview which Montagnier gave to the French Journalist Djamel Tahi in
1997, he stated: "analysis of the proteins of the virus demands mass
production and purification. It is necessary to do that". Indeed, the only
way to prove that a protein is a viral constituent is to obtain it from material
which contains nothing else but viral particles. Instead Montagnier’s and
Gallo's groups incubated the proteins which banded at 1.16 gm/ml with sera from
AIDS patients. The proteins which were found to react more often with the sera
were said to be HIV proteins (although neither group published any evidence for
the existence of retrovirus-like particles, pure or impure, at the 1.16 gm/ml
band); and the antibodies to be HIV antibodies. However, even if the
antigen/antibody reaction is 100% specific, that is, antibodies react only with
inducing antigens and with no other, from such a reaction it is impossible to
determine the origin even of one reactant much less the origin of both the
antigen and the antibody. Let us consider differing scenarios:
nothing else but retrovirus particles and that their antibodies reacted
specifically with their proteins. According to Montagnier, in the cultures
which contain cells originating from patients with adult T4 cell leukaemia,
as did Gallo's HUT-78 cell line, "it is a real soup" of
retroviruses. Indeed, even if the cultures do not harbour HIV they will
harbour HTLV-I and, given the condition used by Gallo and the fact that the
cells were leukaemic, they may also contain endogenous retroviruses.
According to one well known HIV expert, Myron Essex, 35% of AIDS patients
possess antibodies to HTLV-I. (In the same issue of Science in which
Montagnier published his isolation of "HIV", Gallo published three
papers claiming isolation of HTLV-I from AIDS patients suggesting this
retrovirus was the cause of AIDS). According to another equally as well
known HIV expert, Reinhard Kurth, from the Paul-Ehrich Institute in Germany,
70% of "HIV-positive patients" have antibodies which react with
the retrovirus HTDV/HERV-K, an endogenous retrovirus, or, as Kurth put it, a
retrovirus present "in all of us". Thirty seven per cent of HIV
positive individuals were also found to have antibodies to type D
retroviruses whereas HIV is claimed to be a Lentivirus. Although
Montagnier's cultures may not have contained HTLV-I, the result may still
have been "a real soup" of endogenous retroviruses especially if
one considers their culture conditions and the cells used, umbilical cord
lymphocytes, which have been shown to release retrovirus-like particles even
when not infected with HIV. Since we cannot obtain the proteins from each
particle and characterise them, the next best thing is to take the mass of
particles, disrupt their proteins and position them in an electrophoretic
strip according to their molecular weights. Although we know that the
proteins in the strip are retroviral, we have no way of determining which
protein belongs to which retrovirus if more than one retrovirus is present
at the 1.16 gm/ml band. When the proteins are incubated with sera, we may
find some of the proteins react. From such a reaction we will be able to say
that the antibodies and obviously the proteins are viral but we cannot
determine which protein belongs to which virus and by which virus the
antibodies were induced. We will be definitely wrong if we consider such a
reaction proof that the 1.16 gm/ml band: (a) contains only one retrovirus;
(b) the retrovirus is a new exogenous retrovirus, HIV; (c) the proteins are
the HIV proteins; (d) the antibodies are HIV antibodies; (e) these data are
proof that the patient is infected with HIV even if the antibody antigen
reactions are specific.
gm/ml band was composed of retrovirus particles but that the band also
contained non-retroviral material. This material could have been of cellular
origin (cellular constituents also band at 1.16 gm/ml) and may be of
bacterial, fungal and viral origin (constituents of the many infectious
agents other than retroviruses, known to be present in the cultures and the
patients). It is a fact that in the USA, Europe and Australia individuals
with AIDS as well as those at risk have antibodies to many infectious agents
including viruses such as EBV, CMV and hepatitis B virus. Evidence also
exists which shows that individuals with AIDS and those at risk have
circulatory immune complexes, rheumatoid factor, anti-nuclear,
anti-cellular, anti-platelet, anti-red cells, anti-actin, anti-tubulin, and
anti-myosin antibodies. Montagnier himself demonstrated that individuals
with AIDS and those at risk have high levels of antibodies to the ubiquitous
cellular protein actin whose molecular weight is 41,000, as well as to
another ubiquitous protein, myosin, which has two sub-units of molecular
weights, 18,000 and 25,000. Anti-lymphocyte antibodies have been found in
87% of patients who have a positive "HIV" antibody test and their
levels correlate with clinical status. It is also acknowledged that Africans
with AIDS and those at risk are infected with many agents other than HIV.
We know that it is not possible to take a protein from the 1.16 gm/ml mass
and know from which component it originates. Let us then follow the same steps
as Gallo and Montagnier. Take the mass of material banding at the 1.16 gm/ml,
disrupt the proteins and electrophoretically position the proteins in a strip
according to their molecular weights. Using this technique we are unable to
state from which component of the 1.16 gm/ml band mass a protein on the strip
derives. Next we incubate the proteins in the strip with patient sera and
discover that some of the proteins react with antibodies present in the sera.
Even if the antibody/antigen interaction is 100% specific such a reaction does
permit us to define the origin of the proteins. Yet from such a reaction and
without any proof that the antibodies specifically reacted, Montagnier and Gallo
defined the origin of both the proteins and the antibodies as "HIV".
As mentioned, Montagnier found three proteins in the 1.16 gm/ml band which
reacted with antibodies present in his patients’ sera. These were p25, p80 and
p45. Montagnier concluded that his patients were infected with a retrovirus
which "contains a major p25 protein, similar in size to that of HTLV-I",
but made no comment in regard to the p80 protein. Regarding p45 he wrote:
"The 45K [p45] protein may be due to contamination of the virus by cellular
actin".
In 1997 Montagnier said that the protein he detected in 1983 had a molecular
weight of 43,000 and was actin. (The molecular weight of actin is neither 45,000
nor 43,000 but 41,000. However since Montagnier and Gallo determined the
molecular weights of the proteins by their migration in an electrophoretic
strip, and because the migration may be influenced by other factors, for
example, by the protein's charge, it is possible that these slight differences
in the molecular weight are simply the result of experimental variation).
When Gallo and his colleagues used the cellular proteins as antigens in the
WB they reported: "The most prominent reactions were the antigens of the
following molecular weights: 65,000, 60,000, 55,000, 41,000 and 24,000. Antigens
with molecular weights of approximately 88,000, 80,000, 39,000, 32,000, 28,000
and 21,000 gave less prominent reactions…A large protein with a molecular
weight of approximately 130,000 and a protein of 48,000 were also
detected". In another experiment the "antigens from virus purified
from the culture fluids", that is, the 1.16 gm/ml band, were incubated with
different sera. They found an "Extensive" reaction of the AIDS
patients sera with p24 and p41 and concluded: "these molecules are the
major component of the virus-preparation. P24 and p41 may therefore be
considered viral structural proteins…Furthermore, an antigen with a molecular
weight of approximately 110,000 was detected in the virus preparation but was
below limit of detection in the cells. Also, p39 was present in the virus
preparation…Occasionally an additional set of antigens was recognised by a
serum but their relation to the antigens described above is unclear".
Between 1983-87 the detection of antibodies in patient sera which reacted
with p24 or p41 (Montagnier considered reaction with p24 and Gallo with p41 HIV
specific) was considered proof that the patient was infected with HIV. In the
same period of time it became obvious that a significant number of individuals
at no risk of AIDS had antibodies which reacted with these proteins. Since 1987
most of the proteins which Gallo found to react either in the cell extracts or
the 1.16 gm/ml band are now considered HIV proteins and laboratories require the
presence of antibodies which react with more than one protein before the patient
is considered infected with HIV. The number and identity of antibody/protein
(Western blot) bands required vary from continent to continent, from country to
country and even between and within laboratories in the same country. Thus it is
possible for the same patient to be HIV seropositive in New York for example,
but not in Africa or Australia.
Furthermore, for many years evidence exists which shows that p24 is not
"HIV" specific123,124, p41 is the cellular protein actin,58,125,126
p32 is class II histocompatability DR protein126,127 and p120 / p160
are oligomers of p41.128
Even if proof exists that the "HIV" proteins do indeed belong to a
unique exogenous retrovirus, it cannot be assumed that antibodies that react
with them are diagnostic of "HIV" infection. To prove the specificity
of the antibody tests it is mandatory to:
without AIDS must not exclusively be healthy individuals (since they do not
have high levels of antibodies, one would expect few if any reactions) but
include the sick, such as patients with infectious diseases (other than
those which are said to result from HIV infection), those receiving
chemotherapy and those with auto-immune disorders;
isolation;
is, use HIV as a gold standard for the antibody test.
To date nobody has published such studies using any definition of
"HIV" isolation.
The inescapable conclusion is that the antibody/antigen reaction is not HIV
specific and thus cannot be used to prove HIV isolation, no matter how isolation
is defined.
MORE RECENT DEVELOPMENTS
By 1997 some of the best known HIV experts accepted that:
over-represented in comparison to the relative amount in the cell membrane,
whereas others appear to be absent", and that these proteins
"serve as protective immunogens in vaccination experiments".
is frequently prepared by centrifugation through sucrose gradients",
but in none of the studies "the purity of the virus preparation has
been verified". In other words, up till 1997 nobody has published
electron micrographs of the 1.16 gm/ml band to prove that the "purified
virus" contained nothing else but virus particles.
In that year, in Virology, two studies were published, one by a USA
team, principal author Julian Bess, and the other by a Franco-German group,
principal author Pablo Gluschankof, with the first electron micrographs of
"purified HIV". While in the Gluschankof et al studies the EMs
were from the 1.16 gm/ml band, in the Bess et al band they were from
pooled bands. The authors of both studies claimed that their
"purified" material contained some particles with the appearances of
retroviruses and in fact were HIV particles. But they admitted that their
material predominantly contained particles which were not retroviruses but
"budding membrane particles frequently called microvesicles" or
"mock virus". Indeed the caption to the Gluschankof et al
electron micrograph reads, "Purified vesicles from infected H9 cells (a)
and activated PBMC (b) supernatants", not purified HIV. In further
experiments the supernatant from non-infected cultures was also banded in
sucrose gradients. Both groups claimed that the banded material from these
cultures contained only microvesicles, "mock virus" particles, but no
HIV. Both the "HIV" particles and the mock-virus particles possessed
membranes. In the USA study the "HIV-1 particles" were differentiated
from the microvesicles "by the electron dense cores", whereas in the
other study the "HIV" particles were "identified by the
relatively homogenous diameter of about 110 nm, the dense cone-shaped core, and
the "lateral bodies". However, in the arrowed particles which are said
to be HIV it is difficult if not impossible to locate any which have cone-shaped
cores or bilateral, "lateral bodies". In fact no particle in any study
has the principle morphological characteristic of retrovirus, a diameter of
100-120 nM and surface spikes, knobs. In the Franco-German study the average
"HIV" particle diameter is 136 nM and no particle had a diameter less
than 120 nM. In the USA study the corresponding dimensions are 236 nM and 160 nM.
In other words, the American "HIV" is twice the diameter of the
European "HIV", and all other "HIV" particles. The US
authors did not note or explain this discrepancy and "were much more
focused on showing the mixture of particles in the preparations as opposed to
their actual diameters". The diameter of the microvesicles "range in
size from about 50 to 500 nm". Both the "HIV" and the
"mock" virus particles contained RNA. The RNA of the latter had
contained mRNA which is known to be rich in adenine and which, according to
Gallo is specific to retroviral RNA. According to Gluschankof et al,
"The vesicles contain large amounts of protein and nucleic acids which are
unstructured and thus are transparent by electron microscopy", that is why
many, but not all, appear "empty" by electron microscopy, while the
nucleic acids in the "HIV" particles are structured and for this
reason they appear to have an "electron dense cores". However,
according to a leading retrovirologist, John Bader, the core density can be
changed by external conditions, that is, the culture conditions. It is well
known that a structural virus particle or virus-like particle can become
"unstructured" in the presence of reducing agents. The possibility
cannot be excluded then, that the apparent morphological differences between the
two types of particles may be due to nothing more than the difference in the
redox of the microenvironment in which they are assembled, released or both. It
is significant that actin polymerisation (or actin/myosin interaction)
"mediates HIV budding" and release. Evidence also exists that:
different from those in infected cells.
myosin and other cellular proteins (glycoproteins) and many cellular
processes including budding unrelated to release of retrovirus.
polymers in general is regulated by the redox state, oxidation leading to
interaction.
to oxidising agents. In fact, for the detection of "HIV" proteins
and particles the cell cultures must be stimulated (treated with oxidising
agents).
observed.
The minimum absolutely necessary but not sufficient condition to claim that
what are called "HIV-1 particles" are a retrovirus and not cellular
microvesicles is to show that the sucrose density fractions obtained from the
"infected" cells contain proteins which are not present in the same
fractions obtained from non-infected cells. However, Bess et al have
shown this is not the case. The only difference one can see in their
SDS-polyacrylamide gel electrophoresis strips of "purified virus" and
"mock virus" is quantitative, not qualitative. This quantitative
difference may be due to many reasons including the fact that there were
significant differences in the history and the mode of preparation of the
non-infected and "infected" H9 cell cultures, in addition to the
"infection".
A similar finding was reported by the same authors a few years earlier.
However, while in both studies the proteins of molecular weight "near 42
kDa" (42,000) are labelled as "Actin" and "in the 30- to
40-kDa range" as "HLA DR", all the proteins with molecular weight
higher than approximately 42,000 and lower than approximately 30,000 are left
unlabelled in the earlier paper. In the 1997 study, three proteins of molecular
weight lower than 30,000 are labelled as p24CA, p17MA, and
p6/p7NC and are said to represent "major bands of viral
proteins". However, also according to the authors, "these labels were
added when one of the reviewers asked for them. He felt it would help orient
readers when looking at the figure - the reviewer is correct. We did not
determine the identities of the bands in this particular gel".
In their earlier study the researchers from the USA presented further
evidence that the "viral proteins" were nothing more than cellular
proteins. In their efforts to make an HIV vaccine they immunised macaques with,
amongst other antigens, "mock virus", that is, sucrose density banded
material from the supernatants of non-infected H9 cell cultures. After the
initial immunisation the monkeys were given boosters at 4, 8 and 12 weeks. The
animals were then challenged with "SIV" propagated either in H9 cells
or macaque cells. When the WBs obtained after immunisation but before "SIV"
challenge were compared with the WBs post-challenge, it was found that challenge
with "SIV" propagated in macaque cells had some additional bands.
However, the WBs obtained after the challenge with SIV propagated in H9 cells
were identical with the WBs obtained after immunisation but before challenge. In
other words, the protein immunogens in the "virus" were identical with
the immunogens in the "mock virus". Since both the "mock
virus" and "purified" virus contain the same proteins, then all
the particles seen in the banded materials including what the authors of the
1997 Virology papers call "HIV" particles must be cellular
vesicles. Since there is no proof that the banded, "purified virus",
material contains retrovirus proteins and thus retrovirus particles then there
can be no proof that any of the banded RNA is retroviral RNA. When such RNA (or
its cDNA) is used as probes and primers for hybridisation and PCR studies, no
matter what results are obtained, they cannot be considered proof for infection
with a retrovirus, any retrovirus.
In the interview which Montagnier gave to Djamel Tahi he was asked why they
did not publish an electron micrograph of the 1.16 gm/ml band to prove that the
band represented "purified" virus, as they claimed. He replied that
the reason for this was that even after "Roman effort" in their
"purified" virus they could not see any particles with the
"morphology typical of retroviruses. They were very different. Relatively
different". When Montagnier was asked if Gallo isolated HIV he replied:
"I do not believe so". If there were no retrovirus-like particles in
Montagnier’s "purified" virus, then obviously Montagnier and his
colleagues could not claim to have isolated a specific exogenous retrovirus,
HIV.
THE "HIV GENOME"
To claim that the stretch of RNA (cDNA) is the genome of a unique retroviral
particle, HIV, the most basic requirement is proof for the existence of a unique
molecular entity "HIV RNA" ("HIV DNA") that is, a unique
fragment of RNA (DNA) identical in both composition and length in all infected
individuals. The claim that a stretch of RNA (cDNA) is a unique molecular entity
which constitutes the genome of a unique retrovirus can be accepted if and only
if it is shown that the RNA belongs to particles with the morphological,
physical and replicative characteristic of retroviral particles. Proof of this
can only be obtained isolating the particles, that is, by obtaining them
separate from everything else (purifying them). In 1984 both Gallo’s and
Montagnier’s groups reported finding polyadenylated (adenine rich) RNA (poly(A)-RNA)
in the 1.16 gm/ml band material obtained from "infected" cultures. The
RNA was claimed to be HIV RNA that is, the HIV genome, and its complementary
DNA, the HIV provirus. However,
pure "HIV"particles, according to him neither his band nor that
of Gallo’s contained any particles with "morphology typical of
retroviruses";
cells and even at the 1.16 gm/ml band obtained from
"non-infected" cells;
"HIV-RNA" or "HIV-DNA", while the genomes of the most
variable RNA viruses do not differ by more than 1%. The difference between
the human and the chimpanzee genomes is no more than 2% while there is up
to 40% variation between "HIV" genomes;
and since then many other researchers have been unable to prove the
existence of the HIV genome in fresh lymphocytes from AIDS patients. In
1994 Gallo stated "We have never found HIV DNA in the tumour cells of
KS…In fact we have never found HIV DNA in T-cells".
chain reaction (PCR) studies using small fragments of the "HIV"
genome as primers. However even researchers who believe that there is
proof that the HIV primers and probes used in these studies are HIV accept
that the specificity of this assay, using the antibody as a gold standard,
varies between zero and 100 per cent. Even with the PCR nobody has
reported the existence of the full "HIV" genome in the fresh
lymphocytes of even a single AIDS patient.
Furthermore, despite the ample evidence to the contrary, for most
retrovirologists the finding of a novel nucleic acid in a cell can be due to
nothing else but an infectious agent. Half a century has passed since the Nobel
Laureate Barbara McClintock discovered the phenomenon of transposition which can
lead to the appearance of new genotypes and phenotypes. According to McClintock,
the genome can be restructured not only by transposition but also by other means
as well. In her Nobel lecture of 1983, she said, "rapid reorganisation of
genomes may underline some species formation. Our present knowledge would
suggest that these reorganizations originate from some "shock" that
forced the genome to restructure itself in order to overcome a threat to its
survival...Major genomic restructuring most certainly accompanied formation of
new species". The "genomic shock" which leads to the origin of
new species may be "either produced by accidents occurring within the cell
itself, or imposed from without such as virus infections, species crosses,
poisons of various sorts, or even altered surroundings such as those imposed by
tissue culture. We are aware of some of the mishaps affecting DNA and also
of their repair mechanisms, but many others could be difficult to recognize.
Homeostatic adjustments to various accidents would be required if these
accidents occur frequently. Many such mishaps and their adjustments would not be
detected unless some event or observation directed attention to
them...Unquestionably, we will emerge from this revolutionary period with
modified views of components of cells and how they operate, but only however, to
await the emergence of the next revolutionary phase that again will bring
startling changes in concepts".
As we have mentioned elsewhere9,19 an exogenous retrovirus is only
one possible explanation for the finding of novel nucleic acids in AIDS
patients. Other explanations are:
corresponding proviral DNA present in the cellular DNA of uninfected animals
and which is passed from generation to generation vertically (from parents
to offspring via the germ cell line) and which under certain conditions can
be expressed and incorporated into retroviral particles.
recombination and deletion of: (a) endogenous retroviral sequences or (b)
retroviral and cellular sequences or (c) non-retroviral cellular genes.
sequences (transposons) becoming inserted elsewhere in the genome, or by
retroposition, that is, by particular RNA (retrotransposons) first being
transcribed into DNA and then similarly being inserted into the genome.
Retroposition can "use cellular mechanisms for passive retroposition,
as well as retroelements containing reverse transcriptase". The
retroelements may be retrovirus-like elements or nonviral elements. Not only
can retroposition "shape and reshape the eukaryocytic genome in many
different ways" but the nonviral retroelements may be similar to the
retroviral elements.
A basic principle of molecular biology is that the primary sequence of RNA
faithfully reflects the primary sequence of the DNA from which it is
transcribed. However, in the 1980s RNA editing, "broadly defined as a
process that changes the nucleotide sequences of an RNA molecule from that of
the DNA template encoding it", was discovered. In the process a
non-functional transcript can be re-tailored, producing a translatable mRNA, or
modify an already functioning mRNA so that it generates a protein of altered
amino acid sequences. Sometimes editing is so extensive that the majority of
sequences in a mRNA are not genomically encoded but are generated post-transcriptionally
producing the "paradoxical situation of a transcript that lacks sufficient
complementarity to hybridize to its own gene!". According to Nancy Maizels
and Alan Weiner from the Department of Molecular Biophysics and Biochemistry at
Yale University, "the central dogma has survived hard times. The discovery
of reverse transcriptase amended but did not violate the central dogma of how
genes make proteins; introns qualified the conclusion that genes are necessarily
collinear with the proteins they encode; somatic rearrangement of lymphocyte DNA
called stability of eukaryotic genomes into doubt...and catalytic RNA challenged
the pre-eminence of proteins and breathed new life into the ancient RNA
world". However, the discovery of RNA editing "could come close to
dealing it a mortal blow".
Thus the finding of novel RNAs in human cells, especially those of AIDS
patients and those at risk, can no longer be regarded as incontrovertible proof
that the RNA has been exogenously introduced by a putative HIV or any other
infectious agents. That this may be the case has of late been accepted by Luc
Montagnier. In a written testimony dated February 2nd 2000, to the US
House of Representatives Committee on Government Reform, Subcommittee on
National Security, Veterans Affairs and International Relations, in support of
the work of his colleague, Howard B Urnovitz, (Montagnier is on the scientific
advisory board of a publically traded biomedical company whose director is
Urnovitz), Montagnier wrote: "I have reviewed Dr Urnovitz's published
research and the testimony prepared for presentation to this Committee and
strongly advise that future research on Gulf War Syndrome should include the
study of the detected genetic material".
Urnovitz and his colleagues presented evidence of the existence, in Persian
Gulf War veterans, of "novel", "nonviral" RNAs,
"possibly induced by exposure to environmental genotoxins". They
concluded: "The patterns of the occurrence of RPAs [polyribonucleotides] in
the sera of GWVs [Gulf War Veterans] and healthy controls are sufficiently
distinct to suggest possible future diagnostic applications…Our studies of
patients with active multiple myeloma suggest that patients with individual
chronic multifactorial diseases may have unique RPAs in their sera. Validated
tests for such putative surrogate markers may aid in the diagnosis of such
diseases or in the evaluation of responses to therapeutic modalities".
In the same year that Montagnier claimed HIV isolation, 1983, in a paper
entitled "Expression of novel transposon-containing mRNAs in human T
cells" researchers from the USA and Canada reported "Using an HERV-H
LTR probe, 6 and 4.5 kb transcripts were detected by Northern blot analysis
which were induced in normal peripheral T cells after treatment with
phytohaemagglutin" (HERVº human endogenous
retrovirus). Phytohaemagglutin has been and continues to be used not only by
Montagnier but by virtually every retrovirologist who claims proof for HIV
isolation.
Since Montagnier agrees with Urnovitz that novel, nonviral RNAs appear in the
Gulf War Veterans, then why should the existence of novel RNAs:
not the result of the many toxins including genotoxins to which they are
exposed?
RNAs rather than the result of the many toxins including genotoxins to which
both the patients and the cultures are exposed? Especially when both
Montagnier and Gallo accept that HIV cannot be detected in cultures which
are not treated with such toxins (oxidant agents) including PHA?
When hard pressed all the HIV experts will ultimately accept the
non-specificity of retroviral-like particles, reverse transcription and
antibody/antigen reactions. That this is the case is illustrated is exemplified
by the fact that at the beginning of the AIDS era they were the first to report
isolation of HIV from individuals at no risk of AIDS.166-168 In 1985
Weiss and his colleagues reported the isolation of a retrovirus from
mitogenically stimulated T-cell cultures of two patients with common variable
hypogammaglobulinaemia. This retrovirus "was clearly related to HTLV-III/LAV"
(HIV); evidence included positive western blot with AIDS sera and hybridization
with HIV probes.168 In 1984 Montagnier and his colleagues reported
that "activated lymphocytes from a healthy blood donor were spontaneously
releasing a virus similar to LAV1 in culture."169 With the
method presently used for "HIV" isolation (reaction of p24 with
proteins in culture) Schupbach and his associates using cultures of whole blood
reported positive results in 49/60 (82%) of "presumably uninfected but
serologically indeterminate" individuals and in 5/5 (100%) "seronegative
blood donors".170
CONCLUSION
In 1983 Luc Montagnier and his colleagues and in 1984 Robert Gallo and his
colleagues claimed to have proven the existence of HIV by purifying retroviral
particles, that is, by obtaining a mass of particles isolated from everything
else and showing that the particles are infectious. A critical analysis of their
evidence shows that neither group presented proof of isolation of a novel
retrovirus from AIDS patients. The phenomena they interpreted as HIV are all
non-specific and were known to be so long before the AIDS era. In fact, given
the origin of the cells and the culture conditions one would be expect to find
all these phenomena even if the cultures are not infected with a retrovirus. In
1997 Montagnier himself stressed that to prove the existence of a unique
retrovirus purification is absolutely necessary and admitted that he had not
presented such proof, and in his view, neither had Gallo. Recognition of these
facts may prove the first step in solving the problem of AIDS.
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Competing interests: None declared
Competing interests: