PAPADOPULOS-ELEOPULOS ET. AL.
REPLY TO DUESBERG (II)
We gratefully acknowledge Peter Duesberg’s criticisms of our paper "HIV
Isolation: Has it really been achieved?". (1) Before responding it
will be useful to define some terms and objectives.
Virus: A virus has two distinct properties, one physical and
the other behavioural. A virus is a microscopic particle able to generate
exact copies of itself when placed inside a living cell, that is, the particle
Those who espouse the viral theory of AIDS accept that a viral particle,
and not a naked protein or DNA or RNA fragment, is transmitted from person
to person and is both necessary and sufficient to induce the several dozen
laboratory abnormalities and diseases that constitute the clinical AID
Isolation: The essence of isolation is the separation of desired
matter from all other matter not the object of concern.
Isolation of a putative viral particle is necessary to:
(a) document and analyse its constituents;
(b) conduct experiments in order to prove it is infectious and thus a virus;
(c) obtain reagents (proteins and nucleic acids) for diagnostic and other
(d) prove that the pathological effects, if any, are due to the virus and
1. 19 ‘HIV’ genomes
"...the weakest point of the HIV-non-existentialists is their
failure to explain the origin of "19 sequences encompassing the full-length"
10-kb-HIV-1 genome" and "19 full-length HIV genomes"".
(a) Let us repeat that the claim of the existence of "19 sequences
encompassing the full length, 10-kb-HIV-1 genome", "19 full-length
HIV genomes" is not one of our making but that of the HIV experts
we quote. The same experts accept that of the "19 full-length HIV
genomes", no two are the same either in sequence or even in length;
(b) The question we set out to answer in our critique was not what is
the origin of the 19 full-length HIV-1 sequences but does the presently
available data prove that these sequences represent the genome of a unique,
exogenous retrovirus, HIV? The answer, we repeat, is NO.
Nonetheless, although it was not our task to determine the origin of
these sequences, we did present a number of alternative mechanisms that
science offers as a "rational origin for such sequences" in addition
to "viruses or other infectious agents".
2. Odds of assembly
"Remember the odds of coming up with even one nucleotide sequence
of 9150 bp by chance are astronomically low namely, 1 in 49150
which is very close to 0."
It is apparent that we and Peter Duesberg are referring to two entirely
different systems, one completely random and the other heavily biased by
cell and culture conditions. True, the probability of assembling a particular
sequence of RNA (DNA) of 9150 bases randomly selecting each of the
four nucleotides is one in 49150 However, this statistical reasoning
bears no resemblance to how nucleic acid polymers are assembled either
in vivo or in vitro and thus on the probability of finding a
particular unique sequence. That this is the case is best illustrated
by Spiegelman’s minivariant, a 220-nucleotide stretch of RNA of unique
length and sequence which was discussed in our Continuum
paper. The probability of assembling such a unique RNA stretch by chance
is 1 in 4220, also "very close to 0"" yet, under certain
conditions in the laboratory, the Spiegelman minivariant is frequently
produced indicating that the assembly of nucleotides is anything but a
random process. Furthermore, the 19 unique sequences do not have to be
assembled from the four, individual nucleotides. They may result, for example,
by recombination of:
(a) stretches of pre-existing cellular DNA sequences;
(b) stretches of DNA sequences of endogenous retroviruses which form
1% of the cellular DNA, a phenomenon accepted to take place quite frequently
and to result in the assembly of novel genomes. It is also accepted that
the conditions affect the recombination both qualitatively and qantitatively.
It is significant that as far back as 1985 both Gallo and Montagnier
accepted that it is not possible to generate "HIV" and the effects
attributed to it unless the cells are activated (stimulated) and
that this year Chermann and his colleagues showed that the infected
cultures contain fragments of the "HIV genome" but after
PHA stimulation there is an increase in the "full-length genome"
and a concomitant decrease in the fragments. (2) Whatever the odds may
be of obtaining by chance the conditions necessary to generate "even
one nucleotide sequence of 9150 bp", it is certain not 1 in 49150.
3. Viral genome
"The non-HIV-existentialists also fail to realize that available
isolation efforts have already adequately identified the 9150 bases as
the genome of a virus".
ln our extensive search of the HIV literature we could not find even
one reference, (although it is possible we may have missed some), in which
the HIV genome was reported to of 9150 nucleotides. The closest length
was reported Montagnier’s group who, in 1984, reported it to be 9.1 to
9.2 kbases and, in 1985, as 9193 bases. (3,4) lf the 9150 base DNA is the
genome of a virus then an absolutely necessary but not sufficient condition
is that the virus in all infected individuals will have a length of 9150
bases. Yet, two HIV genomes of the same length have yet to be reported.
More importantly, the length of an RNA (DNA) fragment, no matter
how often such a fragment is detected, provides no information regarding
its origin. The only way to prove it belongs to a unique virus is
to isolate a viral particle and demonstrate it has a genome of 9150 bases.
This has not been done and the"available isolation efforts" do
not contain even suggestive evidence let alone proof that a 9150 base long
RNA is a constituent of a particle, any particle much less a viral particle.
4. Koch’s postulate
"In order to isolate a given infectious agent, one needs no
more than to isolate it from all other, possible contaminating, infectious
agents, this is in fact Koch’s second postulate".
At the Xth international Medical Congress held in Berlin in 1890, in
response to the question as to how to obtain irrefutable proof that a bacterium
caused a specific disease, Robert Koch included in his answer the requirement
that "The pathogen must be isolated and bred in adequate numbers in
pure culture." (5) Apart from omitting the second part, "bred
in adequate numbers in pure culture", Peter Duesberg’s definition
of isolation is somewhat obscure. Can a physician for example, claim to
have isolated his patient with hepatitis by placing him in a room with
patients who may have coronary artery disease, fractures or appendicitis,
but none of whom have infectious diseases? ln fact, in 1987, (6) Peter
Duesberg himself defined the second Koch postulate as, "it [the pathogen]
must be isolated from the host and grown in pure culture", that is,
in the absence of "all other, possible contaminating" agents
including non-infectious agents.
5. Re-isolating ‘HIV’
"Montagnier’s original isolate of HIV from extracellular fluids
is an example. Indeed, Montagnier’s isolate appears to meet functional
standards of isolation adequately, because two of the world’s leading retrovirologists,
Robert Gallo of the NIH and Robin Weiss of the Chester Beatty have re-isolated
only HIV from Montagnier’s virus stock. If Montagnier’s virus had been
grossly contaminated by other viruses or microbes those would have been
found by Gallo and Weiss".
There is no evidence in Montagnier’s "original isolate" which
proves isolation of a virus no matter how liberal a definition one applies
to the word "isolation". As far as "functional isolation"
is concerned, suffice it to say:
(a) ln 1983, like Gallo in 1984, Montagnier reported HIV as a "typical
type-C RNA tumor virus" (7) having a characteristic "cylindrical
core". (8) By 1985 it was reported that the nucleotide sequences between
Montagnier’s first HIV isolate, LAV-1 BRU and Gallo’s first isolate, HTLV-IIIB,
"differ by less than 1% overall". (9) Even though Montagnier
had sent supernatant(s) from LAV-1 "infected" culture(s) to the
Gallo laboratory, "with the express understanding that it could be
used for biomedical, biological and molecular biological studies",
neither Montagnier nor Wain-Hobson considered such differences as proving
Gallo’s HTLV-lllB was LAV-1 BRU and in February 1986 wrote, "Thus
there is only a single AIDS retrovirus, and LAV, HTLV-III and ARV represent
different isolates of the same virus" (italics ours). (9)
Indeed, if there "is only a single AIDS retrovirus", a unique
retrovirus, then genomic differences of "less than 1%" should
be the rule, not the exception. However, unexpectedly, not long afterwards
it was discovered that "If you were to test two HIVpositive people
at random and analyse the genetic material of their strains, they would
differ, on average, by about 13 percent". (l0) As a result, the French
accused Gallo of misappropriating their strain which they had sent to him
in 1983. ln other words, Gallo’s isolate of HTLV-IIIB was not a "different
isolate" of HIV but LAV-1 BRU which Gallo transmitted to the permanent
cell line HT (HUT78). At the same time they suggested that HIV-I including
their LAV-1 BRU is not a "typical type-C RNA tumor virus" but
"possibly a lentivirus", that is, a taxonomically distinct retrovirus
containing a conical core. Although there was no proof, this suggestion
was soon accepted as fact by almost everybody apart from Gallo’s group
which for many years insisted that HIV belonged to the same family as HTLV-l
and that it is a type-C particle. Furthermore, as already mentioned, the
length of LAV-1 BRU was reported to be 9.1 to 9.2 kb (9193) while that
of HTLV-lIIB as 9749. (11) By 1991, Gallo et al including
Chermann presented evidence including sequence analysis, which showed "that
Gallo’s IIIB did not come from the Pasteur Institute". (10, 12)
(b) In January 1991 Weiss stated that he "cannot exclude the possibility"
that his isolate, CBL1, is either Montagnier’s LAV-1 BRU or Gallo’s HTLV-IIIB.
The reasons given were:
(i) nucleotide sequences representing 2,443 nucleotides (one quarter
of the "HIV genome") in env, tat, and nef, showed
that CBL1 "has 98.0% amino acids in common with LAV-BRU and 97.8%
with HTLV- IIIB (BH10 clone), whereas the similarity in the same regions
between BH10 and BRU is 98.3%. The tat sequence was most variable,
with 94.2% of the CBL1 sequences identical to both BRU and BH10";
(ii) "...monoclonal antibodies raised against CBL1 gag proteins
do not distinguish between CBL1, BRU and IIIB,’ (13)
(i) the genomic differences reported by Weiss are greater than "the
less than 1 %" differences reported between LAV- 1 BRU and HTLV-IIIB;
(ii) should not the antibodies raised against one strain of HIV react
with all the other strains? If different strains of HIV can be distinguished
by an antibody test then how can one perform HIV antibody tests without
having an antibody test for each strain?
(iii) a few months later other British researchers reported that "CBL-1
and HTLV-IllB show striking differences in their biological properties".
Given the above data it is not possible to claim that Gallo and, Weiss
re-isolated Montagnier’s virus. In fact, the groups do not agree between
themselves as to the crucial questions of whic samples were given and received,
and even less as to which samples were sequenced. (10, 12)
In addition, there are two basic scientific reasons which make it impossible
for Gallo and Weiss to "have re-isolated only IIV fror Montagnier’s
(i) To isolate HTLV-IIIB Gallo used the H9 (HUT78) cell line. However,
evidence exists that the H9 cell line releases retrovirus-like particles
even when not "infected with HIV". (15) Furthermore, the HUT78
cell line was established from a patient with "malignancies of mature
T4 cells", a disease which, according to Gallo, is caused by the exogenous
retrovirus, HTLV-l. indeed, as far back as 1983, he claimed to have shown
that the HUT78 cell line contained HTLV-I proviral sequences. (l6) Weiss
obtained his "CBL-I material" from the leukaemic cell line CEM,
a cell line shown to harbor retrovirus even when not infected with "HIV".
(ii) One aspect on which all HIV experts concur is that gp120 is indispensable
for HIV infectivity. Suffice it to quote from Jay Levy’s and Wain-Hobson’s
most recent papers.
"The likely steps in HIV infection are as follows. The CD4-binding
site on HIV-1 gp120 interacts with the CD4 molecule on the cell surface.
Conformational changes in both the viral envelope and the CD4 receptor
permit the binding of gpl20 to another cell-surface receptor, such as CCR5.
This second attachment brings the viral envelope closer to the cell surface,
allowing interaction between gp41 on the viral envelope and a fusion domain
on the cell surface. HIV fuses with the cell. Subsequently, the viral nucleoid
enters into the cell, most likely by means of other cellular events. Once
this stage is achieved, the cycle of viral replication begins" (18)
(italics ours). "HIV-encoded gp120 recognizes the host-encoded CD4
receptor. This interaction leads to a conformational chage in gp120, allowing
it to bind to a second receptor, CCR-5... At some point to be defined,
the amino acid terminus of gp41 is uncovered allowing penetration of the
host cell membrane and fusion of the viral and host cell membranes. Stripped
of its lipid protection, the capsid complex moves into the cytoplasm, and
reverse transcription is initiated". (19)
We could find no data regarding the type of "material" Weiss
received from Montagnier. The samples received by Gallo "were cell-free
supernatants of LAV cultures". However, as Hans Gelderblom and others
have attested, cell-free viral particles do not contain knobs, (spikes),
that is, gp120. (20, 21) This makes it impossible not only for Gallo to
"have re-isolated only" HIV-l BRU but even to have transmitted
it to his cultures. Given the facts that:
(i) AIDS patients and those at risk are diagnosed as infected with many
agents. These include cytomegalic inclusion virus, Epstein- Barr virus,
herpes simplex virus and
Hepatitis B virus. The latter is present not only in hepatocytes but
like, "HIV", also in T-lymphocytes (22, 23) It is also
accepted that most cultures contain Mycoplasma; (29)
(ii) To "infect" their cultures, Montagnier, Gallo and Weiss
did not use "pure HIV" or even the material which from the cultures
banded in sucrose density gradients at 1.16 gm/ml. but "cell-free"
culture fluids; it would have been a miracle, if they had looked, for
"Montagnier’s virus" to have not "been grossly contaminated
by other viruses or microbes" and for Gallo and Weiss not to have
found such agents irrespective of which strain of "HIV", Montagnier’s
or theirs, they had "re-isolated"
6. All cells have RNA
"...viruses can also be isolated as infectious nucleic acids
from infected cells".
Viruses are not mere nucleic acids. Neither can the introduction of
nucleic acids into cells and their reproduction be considered as proof
for viral infection. lf:
(a) one starts with a presumption, but no proof, that a cell is infected
with a unique retrovirus;
(b) chooses from its RNA a fragment of arbitrary length, and calls it
(c) inserts the RNA (cDNA) into a cell and reproduces the same RNA (cDNA)
and interprets this as infection;
(c) construes (a)-(c) as proof of isolation of a unique retrovirus;
then, given the fact that the same steps can be achieved with any cellular
RNA (DNA), one would have no choice but to consider every single fragment
of cellular RNA (DNA) as retroviral, and that all cells are nothing more
than an assembly of retroviruses.
"...such infectious nucleic acids initiate replication of virus
in uninfected cells from which new virus particles are subsequently released".
This may be the case with the genome of other infectious agents but
this has never been shown for the genome of HIV.
"...infectious HIV DNA has been isolated from infected cells
several times by molecular cloning".
This matter has been discussed at length in our Continuum paper.
Suffice here to stress two points:
Retroviruses are not "cloned, infectious HIV DNA of 9150 bases"
but "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)". (25) Furthermore,
such particles share the physical property of banding at a density of 1.16
gm/ml in sucrose density gradients, a fact long used in their isolation
. Cloning of a virus is defined as obtaining EXACTLY the same virus by
introducing its genome into a cell. However, to date, nobody has reported
such particles by "cloning, infectious HIV DNA of 9150 bases",
or DNA of any other length. In fact, nowhere in the HIV literature can
one find particles which have "a diameter of 100-120 nm" AND
which are "studded with projections (spikes, knobs)", let alone
such particles banding at 1.16 gm/ml in sucrose density gradients. Since
cloning is a process leading to the production of an exact copy of whatever
object one starts with, how can one claim cloning of something before there
is proof that it ever existed?
What does one have to do and how hard does one have to plead in order
to obtain answers to fundamental questions regarding a retrovirus which
has menaced the world and in whose name hundreds of thousands of people
have died or been poisoned?
1. How is it possible to transmit a cell-free retrovirus, "HIV",
when it is accepted that:
(i) gp 120 is absolutely necessary for the virus to enter the cell and
for the "cycle of viral replication to begin";
(ii) to date nobody has reported the existence of cell-free particles with
the dimensions of retroviral particles possessing knobs, that is, gp 120?
2. How can one claim that AIDS patients and those at risk are infected
with a unique retrovirus, HIV, when to date nobody has even reported in
fresh, cultured tissue, or tissue co-cultures, particles fulfilling the
principal morphological and physical characteristics of retroviral particles?
We agree with Peter Duesberg that "the cause that unites us all"
is finding a solution to AIDS. With this our aim we were among the first
to put forward non-infectious factors as agents to explain AIDS in gay
men and furthermore we were the first to propose a non-infectious theory
with a unifying mechanism to explain the development of AIDS in all risk
groups. (26) Indeed, our theory also predicts a non-infectious explanation
for the phenomena which others have inferred as "isolation" of
a novel retrovirus from AIDS patients. However, once HIV was accepted as
the causative agent, we realised that the single most important obstacle
in finding the explanation for AIDS is the belief in HIV. For this reason,
from the beginning and unlike anybody else, we have critically analysed
the data which claim proof for the existence of a unique, exogenous retrovirus,
HIV, in AIDS patients and have always maintained that no such proof exists.
Eleni Papadopulos-Eleopulos, Valendar F. Turner, John M. Papadimitriou
& David Causer
Source: Continuum Feb./March 1997
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