Re: Where are the proper controls in "HIV" research? 20 July 2004
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Nicholas Bennett,
Infectious Disease Postdoc/Clinician
Department of Pediatrics, University Hospital, Syracuse NY

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Re: Re: Where are the proper controls in "HIV" research?

The Perth Group response to my recent post on HIV existence hardly deserves a reply, so poor is it, but I shall oblige anyway.

As to how I know that the majority of scientists agree that HIV RNA and proteins exist, I have to stop myself laughing out loud. Basic science undergraduates are taught some methods used to prove the existence of RNA and protein, and certainly students at a PhD level. Such methods have been applied and are being applied on a daily basis, only not for the purposes the Perth Group expect. Anyone in the field knows what is and isn't accepted: you might as well ask "how do I know that the majority of astrophysicists believe the moon is spherical"?

The Perth Group clearly failed to comprehend the basis of my argument for the infectious molecular clone. The sequence can be translated using the genetic code into 9 open reading frames, and the proteins corresponding to those predicted sequences can be detected in viral cultures and preps. How much more proof can you get?! I have sequenced the viral plasmids, used them to express proteins, then characterised the proteins. I have used the plasmids to transfect cells, then prepared virus and repassaged it, then taken that new virus and sequenced the RNA to obtain 100% identical sequence from what went in. That experiment is not possible without the plasmid being an infectious molecular clone of a virus! EM is not necessary, because the molecular evidence is so strong. EM would be supportive, I'll readly admit, as would analysis of all the proteins, but my methods required the existence of the entire HIV genome in order to work. Others have done far more extensive protein analysis (eg the Richieri paper). I provided extensive evidence for the existence of a molecular clone already: it is clearly their choice whether or not to accept it.

To say that Gallo couldn't find the genome in infected patients is plainly ludicrous, since he was one of the authors of Shaw et al, Science 1984 where they describe cloning of HIV genomes from patient T cells! I was sure that the Perth Group wouldn't misrepresent my words, but clearly I gave them too much credit. Ho et al also compare HIV provirus load with actively replicating infected cells in their 1989 NEJM paper I quoted above. This wouldn't be possible without the HIV genome being present in infected individuals.

They also say that :

"From an antibody-antigen reaction it is not possible to distinguish between "self versus non-self" or to identify the origin of one reactant when the other is known, much less both [unknown]". (edited for presumed meaning and clarity).

I agree it is not 100% certain, but I said it was "a measure of identity". It is rare for an animal to produce auto-antibodies, certainly compared to allo-antibodies. This alone is a clue and should not be rejected out of hand. This is simple common sense, for any practising scientist. Future work (eg passage of this putative virus, showing that the proteins are encoded by the putative RNA) are all confirmatory. The evidence is built up, it doesn't come in one convenient package!

And as for their statement on control cultures:

"There is a very simple reason for this, the control cultures are not stimulated. In fact, one of the many problems in "HIV" research is the lack of controls. In addition, the experiments are not blind".

I plainly gave them enough rope to hang themselves. To say that the control cultures are not stimulated is sheer mindless ignorance! Such experiments would never get published. Controls are everywhere in HIV research, one merely has to bother reading the materials and methods section of papers. When a statement is made like "HIV is found in seropositive people" is it taken as read that "HIV is not found in seronegative people". See the quotes I gave regarding HIV cuture correlating with PCR and antibodies. For every statement there has to be an opposite test (the Perth Group plainly agree with this at least!), and there usually is. Needless to say that my viral passage described above included uninfected cells treated identically to the other cultures, and all protein and RNA analysis was performed on samples as well. One RT-PCR experiment included 8 controls for one experimental sample: and I would have been torn apart by my supervisor for doing anything less.

As for experiments not being blinded, they are clearly confusing the placebo effect in clinical trials with the rather more certain effects of benchtop science (well, effects are certain even if results are not!). Every scientific publication should be rejected under these ludicrous criteria!

The evidence that the RNA encodes the proteins is pure and simple that the sequence can be decoded to the same amino acid sequence as the proteins. I assume that the Perth Group are aware of the genetic code, di -deoxy chain termination and Edmann degradation. The Richieri paper certainly shows that the proteins are viral and confirms their identity by serology, size and protein sequencing. The genetic code itself is deposited in Genbank and is conveniently translated as well. Anyone who has performed well-controlled science will vouch for the correctness of the sequence in the database compared to actual specimens in use.

The fact that the RNA encodes the proteins may not be proof that they are of "HIV", but they are obviously proof of the existence of something exogenous which genetically looks like a complex retrovirus. To say anything else is to deny basic science. To say that the virus is the cause of AIDS, and then call it HIV, further work was done which I summarised in my recent post.

The Perth Group do indeed acknowledge its toxic effect: in their article "ISOLATED FACTS ABOUT HIV - A REPLY" on their website they say that:

"...unlike all other retroviruses, HIV is said to kill cells. Thus, unlike the supernatants (cell free culture fluids) from other retroviral cultures, in "HIV" cultures one would expect to find subcellar material, at least "cellular fragments", microsomes from disrupted cells and "membraneous vesicles which may enclose other cellular constituents including nucleic acids""

Therefore, "unlike all other retroviruses" how can HIV be expected to be purified using outdated methods that were applied to non-cytotoxic retroviruses? Methods like Optiprep centrifugation and Anion Exchange chromatography are required, and yet ignored by the Perth Group both in terms of methodology and results. OptiPrep has been in use for HIV purification for at least 5 years - why do the Perth Group not include it in their isolation criteria?

The quality preps have been mentioned already: others are mentioned below. Any decent "virion-associated cellular protein" paper must use stringent purification methods to prove to the reviewers' satisfaction that the virions are indeed virions and not cellular vesicles.

This paper describes separating HIV from cellular debris.

Dettenhofer M, Yu XF. J Virol. 1999 Feb;73(2):1460-7. Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions.

This one describes RT-PCR of HIV genomes directly from virions.

Mouland et al. J Virol. 2000 Jun;74(12):5441-51. The double- stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation.

Note that the "findings" are not directly related to proving HIV's existance or isolation but the methods used would not be possible if HIV did not exist as a virus that could be isolated from cellular contamination!

It is research like this that ought to unravel any problems with HIV's isolation, but instead it confirms it. Debating half-truths and ignoring facts (and indeed, repeated questions by critics such as whether the Perth Group lied in their analysis of Montagnier's "isolation" paper, or their misrepresentation of viral genetic stability) is not the way to go.

I would feel more sympathy for the Perth Group's criticisms (and I'm all in favour of criticism where it is due!) where it not for the repeated demonstrations of poor literature reviewing, misrepresentation, misunderstandings of basic science and inability to accept information contrary to their hypothesis.

Oh yeah, and what say they regarding Montagnier's RT analysis...? The silence is deafening.

Competing interests: None declared