http://bmj.bmjjournals.com/cgi/elett...7387/495#62150
Re: New and repeated questions and requests to Brian Foley 8 June 2004
Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545
Send response to journal:
Re: Re: New and repeated questions and requests to Brian Foley
Email Brian T Foley
The Perth group wrote:
??
Q1. If the virus preparation is not 100% pure but contains extraneous
elements such as cellular vesicles which contain proteins and
poly(A)-RNA by what means can anyone prove what is cellular and what
is viral? For example, how can anyone determine whether a protein with
molecular weight of 41000 or a poly(A)-RNA present in the impure virus
preparation is cellular or viral?
Note that Brian Foley agreed that the poly(A)-RNA is not specific to
retroviruses and that the antibody-antigen reaction is also
non-specific.
??
The poly-Adenylate tail of an RNA is indeed not specific, but the rest
of the RNA is specific. Likewise although antibodies, even monoclonal
antibodies, are not 100% specific, the specificity of polyclonal
antibodies is closer to 98% specific than it is to 90% specific. The
specificity of monoclonal antibodies can be greater than 99.9%
specific, i.e. a monoclonal antibody can often be shown to bind to
only one protein in a mix of more than 10,000 proteins.
There are textbooks on molecular biology which describe in great
detail the hundreds of different ways that proteins and genes can be
analyzed. It is really not my responsibility to be a one-on-one tutor
for a group of unwilling students who have no interest in the subject
matter, but only want to claim that they have all the answers before
doing any studying themselves.
I might be inclined to be more patient with answering the Perth
group?s repetitious and purposefully ambiguous questions, if they
would answer mine, such as ?Where did you ever get the idea that there
was one set of rules for the purification/characterization of
retroviruses?? or ?Can you show me ONE example of a virus that has
ever met the rules that you think exist??
Because people who understand molecular biology already know that the
Perth group has no clue what they are talking about, and most people
who do not understand molecular biology will never take the time to
read a few textbooks on the subject, I will attempt to explain this in
other terms. DNA is just an information transfer system. It is very
much analogous to the binary code used in computers. DNA without a
cell and proteins to interpret it, is similar to ones and zeroes on a
disk or tape, with no computer to read it. Prior to the mid 1970s
geneticists had figured out that DNA and not proteins, was the genetic
information, but they had little ability to read that information. The
basic method of studying genetics was to induce mutations with X-rays
or other DNA-damaging agents and then attempt to find which defective
processes matched up with damage to which region of a genome. It was
sort of like making scratches on a computer disk and then trying to
find out whether the scratch inactivated Microsoft WORD or Microsoft
EXCEL. Damage to key genes such as those involved in DNA replication
were much more difficult to study, than damage to visible but
non-important traits such as fruit fly eye color. This is analogous to
changing the data in a single cell of a spreadsheet, rather than
changing some part of the operating system or the EXCEL.exe program
itself.
Viruses are very small and very simple organisms. There are viruses
that infect bacteria, called ?phages? and viruses that infect almost
all other life forms. They are so simple that it is very debatable
whether or not they can be considered to be ?alive?. They require
cells and cellular proteins for much of their ability to replicate.
Computer viruses are likewise able to replicate only with the aid of
both computers and other software (usually the operating system, but
sometimes a program such as Microsoft WORD which can replicate viruses
known as macro viruses which use the WORD macros to copy themselves,
or a program such as a mail program to spread a type of virus known as
a worm to addresses found in the mail program?s address bool).
There is nothing really special about either biological viruses or
computer viruses, in terms of the methods used to study them as
compared to the methods used to study the more complex biological
organisms or the more complex computer programs. Biological viruses
move from cell to cell by packaging up their genome in some proteins
into what is known as a virion or viral particle. The ability to
separate the viral particle from the host cell, made them ideal for
studying their genetics, in the early days of genetics before
monoclonal antibodies, DNA sequencing, cloning and other tools became
available. The genetics of the Lambda phage Lac operon was one of the
very first successes in molecular biology. While it took decades of
work by dozens of people working in several different labs to gain a
crude understanding of the Lac operon using those pre-cloning
techniques. Today, a new operon in a newly discovered phage can be
studied and understood in much greater detail by a single graduate
student in a single lab working for 4 years. It?s similar to the
difference between writing a program in BASIC to manage your household
finances on a Comodore 64 computer, vs using QUICKEN or some other
off-the-shelf accounting package on a modern computer.
This ability to obtain a virus genome when it is outside the host
genome was quite critical in the days before cloning made it possible
to obtain any gene or genetic fragment separated from the rest of the
genome. It was similar to being able to obtain a program on a 5 and ?
inch floppy disk, such that it could be moved from one computer to
another, before anyone knew how to use e-mail or wires to move data
from one machine to another. Maybe the use of punch cards would be a
better analogy, as far as historical timing goes. At any rate, just
because floppy disks were ?state of the art? at one time, does not
mean that they are the only acceptable method of moving data from
computer to computer today. Likewise, just because plaque purification
of phages, or sucrose gradient centrifugation of retroviruses was
?state of the art? at one time, does not mean that they are required
methods in the study of phages or retroviruses today.
Asking how it is possible to know that a retrovirus genome is a
retrovirus genome today, is similar to asking how we know that a
string of ones and zeroes on a computer hard disk or attached to an
e-mail message is a computer virus. The short answer is that we have
now seen enough of both of these things that they are easily
recognizable to the people who study them on a daily basis, but they
are still difficult to precisely describe to people who are not in our
fields of study. I am a biological scientist and for me it is simple
to use a few tools such as BLAST to tell me that this:
ccgaagcagg agcagaaaga cagggaacag ggaccgcctt tagtttccct caaatcactc
tttggcaacg acccctggtc acagtaaaaa tagcaggaca gctaaaagaa gctctgttag
atacaggagc agatgataca gtattagaag atataaattt gccaggaaaa tggaaaccaa
aaatgatagg gggaattggg ggttttatca aggtaaaaca gtatgatcaa atacttatag
aaatttgtgg aaaaagggct ataggtacag tattagtagg acctacgcct gtcaacataa
ttggcagaaa tatgttgacc cagattggtt gtactttaaa ttttccaatt agtcctattg
agactgtacc agtagcatta aaaccaggaa tggatggccc aaaggtgaaa caatggccat
tgacagaaga gaaaataaaa gcattaacag aaatttgtac agagatggaa aaggaaggaa
aaatctcaag aattgggcct gaaaatccat acaatactcc aatatttgct ataaagaaaa
Is HIV-1 M group pol gene from a virus that is not easily classified
into one of the M group subtypes.
And that this:
aaggaactag agaggtatta gcaaaaatac caagtaagtt ttgtcctcgt cttcttggta
atccaacttg atttaataaa gccatcgctt cattagcttg cccttcttcc acactgatat
tccataaaac tccttttgca ccaccaccac cttcggaaac tgcttgaact ttattggcat
tgattccttt attggataga aaaacgagta tttcgttagc ttctttttct tctaatccat
ttacaatgac gcgtcttgat tcacagcttg tgaccaaccc catgagagtg attaataaga
aaaattgacg aagaaaataa tacaaatttg tttgtgaaaa agtatttttg gtagtcatga
accgctctgg attaagctta tttttgagag ataattttaa ccagatccta gcagaataat
gaatttagct atagatcttc taagcaaaac aatgaaaatt tctaaagatt cgagggttaa
atagattttg gagttccaga aatacacttt tttcaaatca ttttgcaata aaatttgata
attttaggca aaattcagat gaataatgaa tagccaagat aaaaaagagg ccggattatc
cggcctctga agctgaaaac tcaattactt caaacgatta agctttaatt ttaatgtcta
Is probably part of an elongation factor G gene from a bacteria in the
Chlamydia family.
I am not enough of a computer scientist to know if a string of ones
and zeroes is part of a computer virus or part of a GIF image, but I
have little doubt that there are tools available that can distinguish
between the two. If computer scientists all over the world agreed that
such tools existed and were accurate, I would either just take their
word for it, or begin reading some textbooks and using some tools to
make the determination for myself. I would not try to convince a
government of some African nation that they should stop scanning
internet traffic within their country for a worm or virus string until
there were endless ?debates? about whether or not the global community
of computer scientists can convince me that they know what they are
talking about. Just because I can make clever arguments about computer
viruses needing to be found in the boot sector of 5 and ? inch floppy
disks, does not mean that all over the world computer scientists,
systems administrators, and people who have had there computers
infected by the Netsky virus are lying about the Nestsky computer
virus.
Computer viruses can?t always be found in boot sectors of floppy
disks, and mammalian retroviruses can never be purified to 100% purity
using sucrose gradients. Serology alone or DNA sequencing alone or
protein sequencing alone is not enough to fully classify a new
biological virus, but any two of those three can in most cases provide
all the evidence we need to determine whether or not a virus is
present, and what broad class of virus it belongs to.
Nobody has ever produced an electron micrograph of a computer virus.
Nobody has ever produced and electron micrograph of carbon dioxide or
the human insulin protein. There are many things that human logic can
demonstrate to exist that are not possible to see, even with a good
microscope. Viruses happen to be on the lower edge of the scale of the
sizes of things that are worth looking at. If we could see carbon
dioxide it would probably look just like many other molecules. It is
the chemical properties, molecular mass and other characteristics of
carbon dioxide that are most interesting, rather than what it looks
like. The same holds true for viruses.
The interesting properties of both biological viruses, and computer
viruses, are what they are observed to do to the systems they interact
with. Once we have a computer virus on a disk, or a biological virus
cloned, we can begin to study how it reproduces, and how it affects
the cells or computers that it infects. The fact that there is some
technology involved, that can be difficult to understand if one
becomes confused by arguments about ?specificity?, ?similarity? and
?identity?, does not necessarily mean that the people who use those
technologies are making up lies about them.
The Perth group wrote:
?? Q2. Isn?t it true that to do serological and molecular
characterisation of the virus first one has to obtain its proteins and
genome.
??
No. Cloning a viral genome is so simple with modern technologies that
cloning and sequencing the genome is now the fastest and most
economical route to gain the most information about a viral pathogen,
but it is not ?required?. There are still dozens of other methods that
could be used to characterize a virus. The techniques used to clone
and sequence the first genomes of the SARS coronavirus last year were
not identical to the techniques used with HIV-1 in 1983. Molecular
biology makes advances in technology that are similar to those made in
the field of electronic communications.
The Perth group wrote:
??
We would be grateful if Brian Foley would please give us Levy?s
reference(s) where the complete genome and its genes were determined.
??
Already given above, but here they are again:
Levy JA, Hoffman AD, Kramer SM, Landis JA, Shimabukuro JM, Oshiro LS.
Isolation of lymphocytopathic retroviruses from San Francisco patients
with AIDS. Science. 1984 Aug 24;225(4664):840-2. PMID: 6206563
Luciw PA, Potter SJ, Steimer K, Dina D, Levy JA. Molecular cloning of
AIDS-associated retrovirus. Nature. 1984 Dec 20-1985 Jan
2;312(5996):760-3. PMID: 6096718
Sanchez-Pescador R, Power MD, Barr PJ, Steimer KS, Stempien MM,
Brown-Shimer SL, Gee WW, Renard A, Randolph A, Levy JA, et al.
Nucleotide sequence and expression of an AIDS-associated retrovirus
(ARV-2). Science. 1985 Feb 1;227(4686):484-92. PMID: 2578227
The Perth group wrote:
??
As we stated in our rapid response ?A paraphrased request and a
question to Brian Foley? 14th May 2004, ?We did not ask for the titles
of 90 studies. Especially studies conducted in HIV-2, SIVs, SHIVs, BIV
and FIV. Neither for ?HIV-1? studies which have no evidence for the
existence of ?infectious molecular clones?. Let us paraphrase our
request: Would Brian Foley please give us a summary of the evidence
(not just the title) of a study as well as the evidence from a few
confirmatory studies where the existence of an ?infectious molecular
clone? (as defined by Brian Foley) of ?HIV-1? has been proven.?
??
Yes. I will give a summary of that, right after the Perth group gives
me a summary of any one virus that they feel has been characterized to
their satisfaction. This is not a one-way street, I have the right to
ask a few questions too.
The Perth group wrote:
??
In the Fisher, Gallo et al study, the authors ?used a transfection
technique to investigate the biological properties of molecular cloned
HTLV-III DNA.? The clone used was lambda-HXB2 whose ultimate origin is
a poly(A)-RNA originating from the 1.16g/ml band.
??
This is a blatant lie. The Perth group has been repeatedly informed
that the Lambda-HXB-2 infectious molecular clone of HIV-1 M group
subtype B was NEVER passaged through a sucrose gradient.
The Perth group wrote:
?? (b) ?Expression of the HTLV-III gag-related proteins p15 and p24 by
transfected celled was demonstrated using special monoclonal
antibodies.? That is, by a totally non-specific reaction.
??
That is another blatant lie. Binding between monoclonal antibodies and
proteins is not ?totally nonspecific?. Monoclonal antibodies are VERY
HIGHLY specific, closer to 99.99% specific than to 99.0% specific.
The Perth group wrote:
??
Nowhere in references 14,15 and 16 is there any evidence that the
ultimate origin of the molecular clone, that is, the stretch of DNA
called 93IN101, is an RNA which originated from ?HIV? particles. In
other words, there is no evidence that ultimately 93IN101 is the cDNA
of an RNA found in ?HIV? particles, the ?HIV? genome.
??
The evidence is in there, the Perth group is just not going to admit
that they do not understand the evidence.
The Perth group wrote:
??
Q4. Is It true that the "proviral DNA" is a transcript (reverse) of a
poly(A)-RNA identical to the poly(A)-RNA which, in sucrose gradients,
bands at the density of 1.16g/ml and thus that its ultimate origin is
this RNA.
??
No. Proviral DNA is nearly identical to viral RNA, but because of the
error rate of the reverse transcription process, nearly all integrated
proviral genomes contain one or more mutations relative to the RNA
genome from which they were reverse transcribed. In addition to this,
the viral particles contain two RNA genomes, sometimes derived from
two different proviruses, and the reverse transcriptase can and does
switch templates between these two genomes during the reverse
transcription process. Thus any proviral genome can be a recombinant
between two different viral RNAs.
Proviral genomes are similar, most often very highly similar, such as
99.98% identical, to RNA genomes from the same isolate of virus, but
they are almost never ?identical?.
The Perth group wrote:
??
Q9. Is it true that the molecular clones, ëHXB-2 and ëHXB-3 were
obtained from the ?HIV? provirus and thus that the ultimate origin of
ëHXB-2 is a poly(A)-RNA identical to the poly(A)-RNA from the 1.16g/ml
band.
??
We can never know the answer to that question. Robert Gallo?s lab and
Luc Montagnier?s lab both ended up creating molecular clones of HIV-1
M group subtype B virus that were so nearly identical to each other as
to be indicative of being derived from the very same patient. During
the investigation as to how that could have possibly occurred, many
other clones were made from the patients known as ?LAI? and ?BRU?.
None of them were 100% identical to Lambda-HXB-2, Lamda-HXB-3,
Lambda-BH8 (all from Gallo?s lab) or the LAV-1(BRU) clone (from
Montagnier?s lab), but all of them were far more similar to all of
those clones than to the clones of HIV-1 M group subtype B obtained
from any other patient. See for example:
Guo HG, Chermann JC, Waters D, Hall L, Louie A, Gallo RC,
Streicher H, Reitz MS, Popovic M, Blattner W
Sequence analysis of original HIV-1.
Nature. 1991 Feb 28;349(6312):745-6. No
PMID: 2000145
Both the Montagnier lab group, and the Gallo lab group made many
different preparations of viral RNA. Not all of them came from the
same patient from which the Lambda-HXB-2 clone was derived from. RNA
preparations from viruses derived from that same patient would be on
average 98% or 99% identical to the Lamda-HXB-2 clone, whereas RNA
preparations derived from other people infected with other HIV-1 M
group subtype B viruses in the 1982 to 1984 time period would be on
average closer to 92% identical to the Lambda-HXB-2 clone in the env
gene, and 95% to 97% identical to the Lambda-HXB-2 clone in the pol
gene. For comparison purposes, the pol gene of any lentivirus is
roughly 60% identical to any HTLV-I or HTLV-II virus, which is toward
the lower limit of sequence similarity needed for meaningful
hybridization.
The Perth group wrote:
??
Furthermore, we would be grateful if Brian Foley would please answer
all our previous questions and well as those asked here.
??
For a group that claims to know all about the isolation and
characterization of retroviruses (or lack therof), you have far too
many questions that would be better answered by reading some
textbooks. But if you can give me one example of a virus that you
think has met your criteria for isolation and characterization, I will
illustrate for you how the characterization of various isolates and
clones of HIV-1 have also been done.
Is it just monoclonal antibodies to HIV-1 M group subtype B virus that
you believe are ?totally nonspecific? or do you believe that all
monoclonal antibodies are ?totally nonspecific? despite tens of
thousands of publications to the contrary?
Competing interests: None declared
Absence of evidence is not evidence of absence. 8 June 2004
Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545
Send response to journal:
Re: Absence of evidence is not evidence of absence.
Email Brian T Foley
The Perth group wrote:
??
It is clear that the evidence given in the Fisher et al study does not
demonstrate the existence of an ?HIV-1 infectious molecular clone? as
defined by Brian Foley: ?The clone must produce virus particles that
are identical by serology, morphology, protein sequences, RFLP,
Southern blotting, etc. to the parental virus, and the particles must
also be infectious. If a cloned viral genome does not meet these
criteria, it is not an INFECTIOUS molecular clone of the virus, be it
HIV-1 or any other virus? (his emphasis). (See Brian Foley?s rapid
response ?Re: Re-phrasing our two questions to Brian Foley?, 11th May
2004)
??
I did not state that ALL of those forms of evidence must be researched
and described in each and every paper describing a clone. If I had
meant that all lines of evidence must be researched and presented,
then I would not have used the abbreviation ?etc.? which means that
there are many other lines of evidence that any infectious molecular
clone would also meet IF it were studied.
If an infectious molecular clone of HIV-1 M group subtype B virus is
used to produce viral particles and those particles are studied by EM,
the particles should have the morphology of HIV-1 M group subtype B
viruses produced from other clones, or seen directly in biopsied
tissues of HIV-infected people. This is not to say that the particles
MUST be observed in order to know that the clone is infectious.
If I say that humans have two arms two legs, hair on some body parts
but not ?fur? like many other mammals, etc? It does not mean that if
we can find one human with an amputated leg, that humans do not exist.
Nor does it mean that if a human calls you on the phone to chat, you
have to seriously question whether you are talking to a human because
you cannot see both arms, etc?
Nobody will ever publish a single paper in a scientific journal that
describes all experiments that can ever possibly be done on a virus.
Typically each paper in a peer-reviewed scientific journal makes just
one point or a very few related points. It used to be common to
publish a whole series of papers which in total just reported cloning
a single gene. With modern technologies, cloning is much simpler, so
few papers today even report on the details of the methods used.
As just one example, the 1973 Spectra paper (1) that the Perth group
gets all excited about does not present any evidence that the virus
mixture they were working with was in fact the Moloney-MSV plus
Moloney-MuLV mix. They present no serology, no Southern blots, no DNA
sequence information, no proof that the virus mix causes tumors, no
Western blots showing that the viruses produce proteins of the
expected sizes. The ONLY evidence they present is reverse
transcriptase activity (and even with that, they don?t show that it is
retroviral RT which uses Mg2+ rather than Mn2+), and photos of little
round dots which they claim are EMs from gradient-enriched virus
fractions. This does not mean that we all have to seriously doubt that
Sinoussi et al were in fact working with this particular virus mix. It
only means that if we want to know more about the virus, we need to
look in their METHODS section to see if we can find out more. If we
believe there is evidence of fraud, or if we are just curious if we
can reproduce their results, we can ask Sinoussi et al for samples of
their viruses or cell lines which produce them.
Although the 1973 Spectra paper does not describe every detail of the
Moloney-MSV plus Moloney-MuLV mix of viruses, there are many other
papers that do describe other aspects of this mixture, and they
support the research done by Sinoussi et al, rather than making us
suspect that the Sinoussi paper was fraudulent.
If the Perth group has any evidence that any of the infectious
molecular clones of HIV-1 that have been described are fraudulent,
they should pursue that claim in a serious manner, rather than
"debating" in unmoderated internet forums or trying to gain political
support for their ideas. REFERENCES:
1: Sinoussi F, Mendiola L, Chermann JC, Jasmin C, Raynaud M.
Purification and Partial Differentiation of the Particles of Murine
Sarcoma Virus (M. MSV) According to their Sedimentation Rates in
Sucrose Density Gradients.
Spectra 4:237-243 (1973)
Competing interests: None declared
Uiopp <uislad@faaa.co.nz> wrote in message news:<uislad-AD04EE.01093625062004@lust.ihug.co.nz>...
The entire thing is too large to post here, but it is pretty well
summed up in Nick Bennett's recent addition:
http://bmj.bmjjournals.com/cgi/elett...7387/495#65350
------------------------------------
The statements on retroviral isolation and purification (for various
personal values of "pure") repeatedly made by the Perth Group seem
ironic considering they have no first-hand experience with such
matters.
However, it seems "common sense" that such stringent standards as 100%
pure virus preparations are hardly a prerequisite for discovering new
viral proteins or RNA to a degree sufficient to satisfy most
scientists of their existence. At the very least, I ask the Perth
Group where exactly the sequences deposited in the Genbank database
are supposed to come from? They have the genetic appearance of an
exogenous complex retrovirus and produce virus when innoculated as
plasmids into culture systems. The sequences do not appear in the
human genome. The fact that this is a post -hoc discovery to the
original isolation of the clones should be seen as confirmation,
rather than dismissed because the original findings do not meet
artificial and impractical criteria for isolation.
As an example: Montagnier's original discovery of HIV describes how
proteins from a culture of the putative new virus reacted with sera
from patients with AIDS. In a Western Blot, this not only gives a
measure of identity (self versus non-self) but also a degree of
analysis (size). Such methods are frequently used to show the
prescence of new proteins in a mixture and do not detract from the
question of whether the protein exists or not. The inclusion of
appropriate controls (non-infected samples) neatly sidesteps the
problem of isolation, since proteins and RNA found only in infected
cultures can most likely only come from an exogenous source such as
(for example) a virus in a virus culture. For the record I do not
personally agree with the use of the word "isolation" in that context,
but "detection" seems entirely appropriate and does nothing to detract
from the conclusions of the paper.
One must however, grant that inducible expression of self proteins or
RNA is not a more likely explanation. Previous arguments I have seen
use the appalling explanation of "culture stimulation" to explain
these findings, which I hope will not surface here. One only has to
ask "why does the control culture not show the same 'stimulation'?",
and assume that critics who use such an argument (such as the Perth
Group in their comments on "confounding molecules" of culture
supernatants) are entirely unfamiliar with the practise of using
controls in their experiments. Subsequent work, as I have mentioned,
has shown that the genetic sequences of HIV are not present in the
human genome, so they must be derived from exogenous nucleic acid,
which in the case of HIV is in fact integrated into the host cell
chromosomes. The fact that individual cells within an infected host
display variation in detection of HIV further highlights the fact that
these are not "susceptibility" markers of some other future disease.
As regards the appearance of new proteins, the coincidence of
seropositivity argues against these being inducible host proteins, and
the size can be used as a crude by effective means of identification.
Certainly it is possible to judge with the naked eye whether a
particular Gag protein on an SDS-PAGE gel comes from HIV-1 or HIV-2,
and a pattern of sizes showing approximately 120kD, 55kD, 41kD, 24kD
is suggestive to any retrovirologist that it may show the envelope and
capsid proteins. In addition, the slight cross-reactivity to HTLV sera
described in the early papers increases the likelihood that these
proteins are from a related retrovirus rather than host-derived or
indeed from any other type of pathogen.
And in the end of the day, the presumed HIV RNA actually encodes the
presumed HIV proteins, which in and of itself is a massive clue as to
their mutual identity. One must agree that the situation is beyond
reasonable doubt. Being able to extract a single species of RNA from
the band at 1.16mg/L was always possible, since it was why
retroviruses were originally presumed to have diploid RNA genomes and
how the early work on mapping was performed. It is entirely possible
to reverse-transcribe an entire HIV genome from such a prep, since I
myself have done so albeit using a more crude sample (pellet rather
than density band). The trouble of course is that I wasn't aiming to
answer the Perth Group's questions on isolation, merely perform
site-directed mutagenesis - the point being that the field has moved
on beyond trying to address such questions.
The Perth Group appear fixated with wanting a quick-fix (i.e. one
paper) comprehensive answer to their questions, which as far as I am
aware is not possible using historically relevant data. Much of the
data directly answering their questions was published after the
acceptance of the existance of HIV and its causal link to AIDS - in
particular I refer them to my earlier reference using anion-exchange
chromatography, since as well as providing an excellent EM the protein
content of the virions was also extensively analysed. Since they seem
well practised in pulling together disparate facts from papers, I urge
them to undertake a systematic review of the literature with a view to
understanding why the conclusions about HIV's existence and
pathogenesis have been made, rather than trying to support a tired and
well-refuted argument. If they desire a comprehensive review then I
suggest they indulge themselves with a little light reading of Field's
Virology.
My point is that within the limitations of the science at the time,
the approach used was entirely reasonable. One must bear in mind that
many of the quotes used by the Perth Group refer not to complex
cytotoxic lentiviruses but to comments and suggestions made with
reference to simple non-cytotoxic oncoviruses such as RSV, ALV etc.
These viruses are well known to produce far better quality preps than
HIV due to the nature of HIV to kill cells in culture and produce a
soup of cellular debris. This toxic effect has at least been
acknowledged by the Perth Group, which makes their insistence of
requiring such soup to have been removed from the very first analyses
(of these unknown, at the time uncharacterised virus preps) all the
more surprising. Anyone familiar with the practical problems of
benchwork would acknowledge the difficulty of attaining perfection in
the very early days of any line of investigation.
In addition, the later quotes will include assumptions of using
refinements to the techniques of isolation and purification far and
away superior to the mere density banding at 1.16g/ml in a sucrose
gradient. It is entirely possible to directly address the problems of
viral versus host protein and RNA in a prep using modern methods, and
the fact that these findings are retrospective does nothing to detract
from the original work. Science works by progression, adaptation,
correction and augmentation of knowledge. Many of the original
assumptions and conclusions about HIV (and many other pathogens) have
been found to be wrong, but the actual existence of the virus and its
ability to replicate has, if anything, been more firmly established
than ever before.
Additionally, I should point out that having worked with HIV-1 and
HIV-2, and with other researchers with the same and far greater
experience, I believe that my opinions are in line with the vast
majority of those working in the field, including those of Brian Foley
and Christopher Noble. That's not to say that purification and
isolation are not goals to be attained, but a huge amount of work can
be done in their abscence including, but not limited to, detection,
proof of exogenous existence, and causal links to disease processes.
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