Response to Gregory Benvenuti 3 December 2004
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Nicholas Bennett,
Infectious Disease Postdoc/Clinician
Department of Pediatrics, University Hospital, Syracuse NY

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Re: Response to Gregory Benvenuti

I appreciate Mr Benvenuti's moderated questions. It makes a pleasant change from some of the more antagonistic and dogmatic statements made without any support.

The story he describes is an approximation of that of HIV, but not entirely true. For instance, retroviruses had up until then been largely implicated in cancer in animals and birds, rather than immune deficiencies (even though some immune suppressive effects are seen). The rough steps taken though are reasonably correct.

It is true that by just measuring "a reaction" between some serum and an antigen mix, you cannot be sure what exactly it is that is reacting. One way to help differentiate this is to compare the reaction between serum and non-virus (low RT) preps and virus-containing (high RT) preps. The mirror would be to compare serum from healthy people against both virus and non-virus preps. It is clear that, with rare exceptions, virus preps only react with serum from the ill patients (and in the early days, prior to the development of good tests, not even all the AIDS patients demonstrated immune responses to the limited test samples).

Another way is the Western Blot, since retroviruses have proteins of particular sizes. This assay not only allows you to say whether a reaction is occurring or not, but also allows you to make a reasonable guess at the identity of the proteins. Here more than anywhere else, you can run non-viral or uninfected samples and demonstrate that the reaction is or is not likely to be viral in nature. Also, including non-viral samples and using healthy serum as an additional control one can identify where false-positive reactions may be contributing to "viral" bands.

The rabbit example is answered by the fact that controls are always part of this kind of research, and if they're missing the paper is generally refused publication. Some kind of control must be done to either confirm the identity of the antibodies formed, and/or include fake innoculations using non-viral preps (presumably still containing the cellular debris also co-purifying in the sucrose gradient).

While the association cannot of course prove 100% causation, our knowledge of antibody/antigen reactions tells us that this is highly unlikely - antibodies are perhaps second only to nucleic acid hybridisations as far as their specificity is concerned.

There are EM's of virus particles out there: Larry Arthur has published several and an oft-quoted paper by Richieri also has pictures. The early problems with contamination can be (and have been) circumvented by additional purification steps above and beyond those used in traditional retrovirology. Techniques like this have indeed shown that the "real thing" doesn't in fact include all viral proteins (some are expressed but not packaged) and also that virus particles do include some cellular proteins (Cyclophillin A, Staufen) which have been strongly implicated in allowing the virus to correctly replicate. Virus genomes have been sequenced from these preps, and it is relatively easy to check to see how much non-viral RNA is present in a viral prep. This demonstrates that non-viral RNAs are exceedingly rare in a normal prep, even though viral RNAs are rare in the actual cell. This is all very strong evidence indeed that the purification methods are appropriate.

You would not need to necessarily prove that the viral particles were "on their own" by EM if you could do so using genetic techniques. There is absolutely no known rationale for explaining why a particular 9.7kb strand of RNA should co-purify with the proteins it encodes, in particular when the purification method was based on selection for a structure vastly larger than the RNA or protein and did not use any pro-RNA or pro-protein selection methods. The only reasonable explanation is that they are contained within a particle such as a virion. Finding something like that would be like discovering a car which had a robot on the back seat programmed and capable of building the car, when provided with sufficient materials. That's all a virus particle really is: instructions for building a construct that itself contains the instructions (and if you believe Dawkins, that all any of us really are!). When you find a small construct that fulfills that criteria, you have a virus and no argument of "contamination" makes sense. it doesn't matter if you also find a Haynes manual in the glove box, or that the robot is also programmed to produce a tyre inflator that isn't anywhere to be found - those are lines of future research rather than reasons to reject the conclusion that the robot built the car. Additionally, future detection of the robot (or indeed, the circuit board from the left arm of the robot) makes it reasonable to assume that the rest of the car isn't far away. This is especially true if when you "purify" this thing by, say, selecting for the rubber on the tyres, you can create a whole new batch of little cars and robots in a culture of scrap metal. The fact that you've also co-purified a bunch of trucks, vans and spare tyres doesn't matter, because they won't replicate. If they did you'd have some explaining to do!

The trick is that RNA molecules like the HIV genome (capped mRNA's with poly(A) tails) typically only encode a single open reading frame. They can make one protein, and one protein only. The RNA of HIV not only theoretically has 9 open reading frames in it (actually 11, since two are split up), every single one of these is active. Superficially it looks like a normal gene, but closer inspection shows it to be far, far unlike anything in the animal world. Certainly no human gene comes close. To find this amazing RNA co-purifying with the proteins it encodes...it's astonishing. The disease association is not just that of a particular nucleic acid sequence, but a sequence unique to an object that appears to be an exogenous retrovirus. Again, our knowledge of such findings tells us that this is strongly suggestive of the role of the actual agent in causing disease. This isn't an a priori assumption from the mere fact we found this sequence, but an extrapolation from past experiences - backed up by various controls and checks (e.g. that the sequence isn't endogenous and part of the human genome, that HIV negative people don't also contain it, that kind of thing).

QT-PCR (quantative PCR) is obviously important in HIV management, because of the association between viral load and disease. The important point is that high viral loads predict _future_ risk of disease, rather than correlating with _current_ risk of disease (CD4 counts are much better at doing that). This also fits with our understanding that the higher the replication and total body load of a pathogen, the faster the disease may progress. As you may well appreciate, HIV is very hard to visualise from the peripheral blood - it hasn't been done without some form of intermediate culture. The viral loads are of course only viral RNAs, and don't necessarily refer to infectious particles. Several lines of research have shown that HIV probably only has a single infectious virus for every 60,000 particles. That's a low number, but not too unusual in the virus world. The so-called "particle to infectivity ratio" is often in the thousands, somtimes in the hundreds. HIV just seems particularly poor: it may be that in its natural state (harmlessly infecting chimps rather than killing humans) it is spread more through cell-to-cell contact (this is seen in chimps infected with HIV, and with HTLV in humans). To argue that the RNA has nothing to do with the syndrome you have to explain the correlations between RNA level and risk of progression: and indeed where the RNA is coming from! Some have argued that the immune deficiency allows greater replication of the virus, hence higher viral load, but of course high viral loads precede the immune deficiency rather than the other way around!

Not being able to count the HIV by PCR wasn't really a problem for HIV researchers. Cultures could be used to measure virus levels to a certain degree, and attempts were made to look at p24 antigen as well (not very successfully). But by the standards of the time, culture and serology was plenty to conclude that the virus was present. PCR was invented in 1983 and by 1986 was already being used to "purify" genetic sequences without actually, er, purifying anything (i.e. you performed the reaction on mushed up tissue samples and relied on the intrinsic specificity of the reaction to detect and amplify only the sequence you wanted). What molecular techniques have perhaps permitted more than anything else is laziness! "Why purify when you can PCR?" should perhaps be the advertisement - and indeed, one could argue that there is no need to persist in trying to EM the virus from peripheral blood if you can "pull out" (even that phrase implies purification) the genome in its entirety from infected cells. Remember that these techniques were not invented to prove HIV exists or causes AIDS, but were merely used as tools along the route. The "mushed up" samples I refered to above were described by Mullis himself in detecting human genes by PCR.

The nutrition issue is a good one: it is certainly true that very chronic and poor malnutrition will result in a lowered immunity. It's also true that this doesn't behave epidemiologically like an infectious disease, whereas AIDS does. The drop in antioxidants is related to HIV infection, and appears to be part of the disease process (no evidence to date suggests that it predisposes an individual to acquiring HIV or an AIDS-defining infection). I agree that the benefits of multivitamins warrents clinical attention in those with HIV. AZT toxicity is bad, but grossly overstated by those keen to stand in opposition to it (not the least of which is the story that it is a too-toxic anti-cancer drug, whereas in truth it was inactive - and AZT is only one part of a large array of antivirals in use today). AZT toxicity during the days of monotherapy was common however, but at doses twice that in use today!

The problem with putting "mutated viruses" in quotes, as if it's an assumption, is that such mutations are not just detected in research papers, but in everyday clinics as part of patient monitoring. They match with the drugs in use, as the virus mutates around them just like bacteria do with antibiotics. For sure some people will have died due to drug toxicities, but the studies suggest that far more would have died had they not been on the drugs.

Ultimately, nutritional status and sanitation (ESPECIALLY sanitation) boil down to a person's ability to fight off an infectious agent. It does not mean that because clean water prevents cholera that vibrio cholerae isn't responsible for the disease! I don't see everyone in the medical establishment trying to pin things on an infectious agent, only those diseases that are appear to be infectious. As well as improved living conditions, some might say that being able to eradicate infection from people (with antibiotics) and prevent infection (with vaccination) was also a rather important factor in reducing levels of TB.

I can't quite see where Mr Benvenuti sees that "The truth is that more and more information is coming to light that is indicating that this [HIV] was a mistake". If anything more and more of the original arguments against HIV (can't be isolated, can't be grown, treatment doesn't improve health) are being refuted - not that they meant much in the first place. The internet has given some opponents to the prevailing view a voice, and caused people to question the view based upon what they have read. The truth is though that only half the story is ever told in these arguments, and the background to how the prevailing view was reached is never seen.

I suppose in summary the best explanation for why the conclusions were drawn that lead to HIV being held responsible for AIDS, are that they were based on previous experience and logic and ultilised very powerful techniques that made other, perhaps more traditional methods redundant.

Nick Bennett njb35@cantab.net

Competing interests: None declared