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THE ISOLATION OF HIV:
HAS IT REALLY BEEN ACHIEVED? Part 3 Note: The information on this website is presented for
educational purposes and This document was provided by Previous: The Isolation Of Hiv: Has It Really Been Achieved? Part 2 6. "HIV DNA" In debating the proof for the existence of a unique, exogenous retro-viral agent one cannot adopt as an initial premise ("Full-length HIV-1 and HIV-2 DNAs...") that is contingent upon proof of the argument ("ergo...HIV exists and has been isolated"). The a priori designation of a particular fragment of DNA as "HIV DNA" merely begs the question under consideration. 6.1 MINIMUM EVIDENCE REQUIRED TO PROVE THE EXISTENCE OF HIV DNA If "HIV DNA" is the genome of a unique retroviral particle then the most basic requirement is proof for the existence of a unique molecular entity "HIV DNA", that is, unique fragments of 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 a particle with the morphological, physical and replicative characteristics of a retroviral particle. Proof of these properties can only by obtained by isolating the putative viral particles, that is, by obtaining them separated from everything else, extracting the nucleic acids and demonstrating that such particles are identical (their constituents including their nucleic acids are identical) and infectious. The correct procedures, now having been used for over half a century to achieve this proof, require demonstration that: 1. In "infected" cell cultures (cocultures) there are particles with a diameter of 100-120nM containing "condensed inner bodies (cores)" and surfaces "studded with projections (spikes, knobs)"; 82 2. In sucrose density gradients the particles band at a density of 1.16 gm/ml; 3. At the density of 1.16 gm/ml there is nothing else but particles with the morphological characteristics of retroviral particles; 4. The particles contain only RNA and not DNA and that the RNA consistently has the same length (number of bases) and composition no matter how many times the experiment is repeated; 5. When the particles are introduced into secondary cultures, but mindful of the critical caveat discussed below:
6. As a result of 5 the cells in the secondary cultures release particles into the culture medium; 7. The particles released in the secondary cultures have exactly the same characteristics as the original particles, that is, they must have identical morphology, band at 1.16 gm/ml and contain the same RNA and proteins. The caveat is that while the introduction of the majority of infectious particles into cell cultures and subsequent release of similar particles is proof that such particles are indeed infectious, this is not the sufficient case for retroviruses. The basis of this exception is the fact that "one of the most striking features that distinguishes retroviruses from all other animal viruses is the presence in the chromosomes of normal uninfected cells, of genomes closely related to, or identical with, those of infectious viruses". 83 In fact, a cell may contain the genome of many retroviruses. As far back as 1976 retrovirologists recognized that "the failure to isolate endogenous viruses from certain species may reflect the limitations of in vitro cocultivation techniques". 84 In other words, the finding of a retrovirus in both the primary and secondary "infected" cultures/cocultures is not proof that the cells have been infected with an exogenous retrovirus. One way which will suggest but will not prove that the cells acquired virus from the outside (exogenously acquired retrovirus, infectious retrovirus) and have not assembled a retrovirus HIV from information already existing in normal cells (endogenous retrovirus) is to conduct experiments that use controls, that is, to run in parallel with test cultures/cocultures control cultures/cocultures. The only difference between the test and control cultures should be the introduction of particles into the test cultures. In other words, apart from the introduction of particles, in every other respect control cultures must be dealt with identically. For example: (a) 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 those of AIDS patients; (b) because the mere act of co-cultivation alone may lead to release of endogenous retroviral particles, if test cells are cocultured, so should the cells used in control experiments; 85 (c) extracts, even from normal unstimulated cells when added to the cultures may increase endogenous retroviral expression. 86 Because of this, when cells are cultured with "HIV" (supernatant or material which bands at 1.16 gm/ml), the controls must be cultured with similar material from cell cultures originating from sick individuals with illnesses similar to AIDS, that is, matched individuals who are immunosuppressed; (d) the appearance of endogenous retrovirus can be accelerated and the yield increased a million fold by stimulating the cultures with mitogens, 87 mutagens, chemical carcinogens and radiation. 88, 89 If test cultures are exposed to or employ such agents so should the controls; (e) since AIDS patients and those at risk of developing the syndrome are exposed to strong oxidizing agents, 79,90 the control cells should also originate from such patients; (g) to avoid observer bias and in the best interests of science, blind examination of test and control cultures/cocultures should be performed. 6.2 EVIDENCE FOR THE EXISTENCE OF "HIV DNA" 6.2.1 In 1984, in the first of two papers, Montagnier and his colleagues described the following experiment: "Because LAV can induce T-cell fusion and because EBV [Epstein Barr virus] is known to have fusion activity in B cells, we performed co-infection experiments of unfractionated lymphocytes (B and T) with both viruses. It was hoped that stable hybrids of LAV-infected T cells and of EBV-transformed B cells would be formed and that such hybrids would be able to continuously produce LAV. Several regimens were tried. The one that gave rise to continuous productive infection of LAV was the following. Whole lymphocytes of F. R. were first stimulated for 24 hours with Protein A and then infected with and EBV strain, M81, derived from a nasopharyngeal carcinoma. Five days later, half of this culture was infected with LAV as described (1) and then divided in two subcultures: one was cultured in medium lacking T-cell growth factor (TCGF: interleukin-2), the other in medium containing TCGF. As expected, the TCGF-fed culture produced LAV as detected by a peak of RT activity appearing between day 12 (day 6 after LAV infection) and day 21 in the supernatant. In contrast, the cells cultured in the absence of TCGF did not yield any detectable RT...On day 19, at the time of decline of LAV production, a subculture of the TCGF-fed cells received fresh T cells from the same donor: these T cells had been activated for three days with phytohemagglutinin (PHA)...Six days later (day 25), a new peak of RT appeared, but contrary to the first infection, it was not transient...At the time of the second LAV infection, large round cells transformed by EBV could be readily seen in this culture, as well as in the control culture not infected with LAV, indicating that immortalization of the B cells by EBV had already occurred. The immortalized B-cell line was termed RF8". 29 [Reference 1 to which Montagnier refers is the 1983 paper in which Montagnier et al described the first "isolation" of HIV (see 5)]. In the second study, 200 ml of supernatant from the "HIV infected" FR8 cells were banded in sucrose gradients, "Virus containing fractions were pooled" and centrifuged. (It is not stated how they deter-mined the existence of "virus", in which band(s) (fraction(s)) "virus" was found, how many bands if any were found to have particles, or why there were more bands than one (1.16 gm/ml) containing the "virus"). The pellet was incubated with several substances, dATP, dGTP, dTPP, dCTP including 32 dCTP and an oligo(dT) primer. From the cDNAs thus obtained, three clones "pLAV13, 75 and 82, carrying inserts of 2.5, 0.6 and 0.8 kilobases (kb), respectively, were characterized further. All three inserts have a common restriction pattern at one end, indicative of a common priming site". "The 50-base pair (bp) common HindIII-Pst I fragment was sequenced and shown to contain an oligo(dA) stretch preceding the cloning dC tail. The clones are thus copies of the 3’ end of a poly(A) RNA. The specificity of pLAV13 was determined in a series of filter hybridization experiments using nick-translated pLAV13 insert as a probe". Firstly, "using an adapted spot-blot technique" they tested the pellet obtained from the supernatant of "LAV-infected" normal lymphocytes and CEM cells as well as non-infected lymphocytes. The "infected" pellets were positive and the non-infected negative. "Second, the probe detected DNA in the Southern blots of LAV-infected T lymphocytes and CEM cells. No hybridization was detected in DNA from uninfected lymphocytes or from normal liver". No details are given regarding the method used to produce "infection", but it would appear that the normal cells and the CEM cells were cultured with supernatant from the FR8 cells, that is, the same supernatant they used to obtain the probe! They concluded: "Together, these data show that LAV pLAV13 DNA is exogenous to the human genome and detects both RNA and integrated DNA forms, derived from LAV-infected cells. Thus, pLAV13 is LAV specific". 91 6.2.2 In May 1984, Gallo and his colleagues published four papers. To "isolate" HIV they used a leukaemic cell line which they called HT. It is impossible to know with what tissues from AIDS patients this cell line was cultured. Reading the May 1984 papers one gets the impression that the HT cell line was cultured with concentrated (supernatant) fluids originating from individual, AIDS patient, stimulated T-cell cultures. Subsequently, the Gallo investigation found the HT cell line was cultured with concentrated fluids pooled initially from individual cultures of three patients and ultimately from the individual cultures of ten patients. 92 The Gallo investigation found this procedure to be "of dubious scientific rigor". One scientist described the procedure as "really crazy". 93 In 1985, Gallo and his colleagues wrote, "The H9/HTLV-IIIB cell line was derived from the human T-cell line HT, following co-culture with T lymphocytes obtained from several AIDS patients, and contains many different HTLV-III forms". 94 The detection of reverse transcription of A(n).dT15 in the supernatant, was considered proof the HT cells were infected with a retro-virus, HIV, which originated from the patients’ tissues. A clone, H9 of the HT cell line was obtained "using irradiated blood of a healthy donor as a feeder". 21 The H9 cells were cultured with supernatant from the "HIV" infected HT cells. The H9 supernatant was banded in sucrose density gradients and the material which banded at 1.16 gm/ml which, without proof, Gallo and his colleagues considered to be synonymous with retroviral particles, was "lysed with sodium dodecyl sulfate (SDS), digested with proteinase K, and directly chromatographed on an oligo(dT) cellulose column. The resulting polyadenylate [poly(A)]-containing RNA was used as template to synthesize 32 P-labelled complementary DNA (cDNA) in the presence of oligo(dT) primers. The size of the resultant cDNA ranged from 0.1 to 10 kb. When these labelled cDNAs were hybridized to poly(A)-containing RNA purified from infected [that is, cells cultured with the same supernatants as those from which the probe was obtained] and uninfected H9 cells as well as other uninfected human cell lines, only the infected H9 cells contained homologous RNA sequences as evidenced by discrete RNA bands after Northern hybridization. Figure 1 shows that cDNA preparations from HTLV-III B and HTLV-III Z gave identical patterns, detecting species of about 9.0, 4.2, and 2.0 kb...These bands are similar in size to those corresponding to genomic size messenger RNA (mRNA) and spliced mRNAs of env and pX sequences previously observed in cells infected with HTLV-I, consistent with the anticipated relatedness of these viruses. Furthermore, viral mRNA bands of HTLV-II-infected cells were detected with an HTLV-III cDNA probe and again the sizes of the mRNA were like those with HTLV-I"! 56 In another study by Gallo and colleagues, extrachromosomal DNA of "infected" H9 cells was extracted and "assayed for its content of unintegrated viral DNA" using the 32 P-labelled cDNA as a viral probe. "Unintegrated linear viral DNA was first detected after 10 hr [of "infection"] and was also present at the subsequent time points. Figure 1 shows a Southern blot of the 15-hr sampling. A band of 10 kilobases (kb) in the undigested DNA represents the linear form of unintegrated HTLV-III". 95 In yet another study Gallo and his colleagues reported that, "Since the HTLV-III provirus was found to lack Xba I restriction sites, a genomic library was constructed by using Xba I-digested H9/HTLV-III DNA, and this was screened with an HTLV-III cDNA probe to obtain molecular clones of full length integrated provirus with flanking cellular sequences. Fourteen such clones were obtained from an enriched library of 10 6 recombinant phage, and two of these were plaque-purified and characterized. Figure 1 illustrates the restriction maps of these two clones, designated lHXB-2 and lHXB-3. The overall length of the HTLV-III provirus is approximately 10 kilo-bases... To determine whether the HTLV-III genome contains sequences homologous to normal human DNA, the viral insert of lXB-2... was isolated, nick translated and used to probe HTLV-III-infected and uninfected cellular DNA. Under standard conditions of hybridization... this probe hybridized to DNA from H9/HTLV-III cells as well as other HTLV-III-infected cells, but not to DNA from uninfected H9 cells, uninfected HT cells (the parent line from which H9 was cloned), or normal human tissues (data not shown). This finding is in agreement with the results of other experiments in which the unintegrated (replicative intermediate) form of HTLV-III was used as a probe and demonstrates that HTLV-III, is an exogenous retrovirus lacking nucleic acid sequences derived from human DNA". 96 6.2.3 In 1984, Levy and his colleagues cultured PBMC from patients suffering from Kaposi's sarcoma with IL-2, polybrene and PHA. The supernatant was tested for RT, the cells for reaction with serum from the Pasteur Institute patient BRU and "some cultures were examined for virus by electron microscopy". The finding of a positive result with "any of these tests" was considered proof of virus isolation. The supernatant from one of these cultures was "inoculated into fresh human PMC stimulated 3 days before with phytohemagglutinin". Within 6 days the supernatant of this culture had high RT activity and this was said to represent "the virus isolate ARV-2". 97 The HUT78 cell line was cultured with "ARV-2". In the HUT78 "Virus production was monitored by measuring reverse transcriptase activity". When there was maximum RT activity, the supernatant was centrifuged and the resuspended pellet, after treatment with DNAase, was centrifuged in sucrose gradients. The nucleic acid from each fraction was electrophoresed on agarose gel. The region in the gel containing an "9kb RNA species was cut out" and used to obtain "a radioactive cDNA probe". The DNA from the HUT78 cell line cultured with "ARV-2" was digested with restriction enzymes, electrophoresed in agarose gel and Southern blotted using the "radioactive cDNA probe". "No specific bands were detected in several digests of DNA from uninfected cells...whereas bands were seen in infected cells...undigested DNA from infected cells contained a species at 5.5 kb, a faint species at 6kb and a broad band at the exclusion limit of the gel (>15kb). We suggest that the DNA species 5.5kb and 6kb represent unintegrated viral DNA in a circular configuration containing respectively one and two long terminal repeats (LTRs); the upper broad band (>15kb) rep-resents provirus integrated into the host cell DNA". In an additional experiment "whole-cell DNA from cells infected with ARV-2 was partially digested with E CO RI; 9-15 kb cell DNA was cloned into an EMBL-4 bacteriophage l vector and recombinant phage were identified with the virus-specific cDNA probe". Among the recombinant phage obtained were l-9B and l-7A, each of which was 9.5 kb. 98 6.2.4 SUMMARY AND DISCUSSION It is obvious that although Montagnier, Gallo and Levy and their respective colleagues refer to virion or virus particles purification or isolation, none of these groups have presented evidence for the isolation of retrovirus particles or even the isolation of virus-like particles, the first and absolutely necessary step in proving the existence of a retroviral genome. (At the time of writing, neither has any other group of HIV/AIDS researchers). Finding some RNA which bands at 1.16 gm/ml, selecting from it a poly(A) rich fraction, or a fragment of a given length, even if always found to be the same length and sequence, and referring to it as HTLV-III, LAV, ARV does not constitute such proof. It must be stressed that even if the RNA is incorporated in a particle which in sucrose density gradients bands at 1.16 gm/ml, this is still not proof that it is retroviral RNA. According to John Coffin, one of the best known experts on the retroviral genome, there are particles "with a full complement of viral proteins, but the particles contain a collection of cellular RNAs and only about 1% genomic RNA...assembly of particles does not require the genome...in its absence other RNA molecules may be substituted". 83 It is important to note that although all groups, Montagnier’s, Gallo’s and Levy’s refer to the material from the culture supernatants which in sucrose density gradients bands at 1.16 gm/ml as viral particles, virions, and to the RNA and proteins at that density as "particle-associated" RNA or proteins, not one of the groups presented evidence for the existence at this density of any particles, retroviral-like or otherwise, pure (isolated) or otherwise. Instead these researchers cultured lymphocytes from AIDS patients and stimulated (activated) them with a wide variety of agents. Reverse transcription of A(n).dT15 in the culture supernatant was considered proof for infection with a retrovirus or even proof of isolation. Supernatants from these cultures were introduced into cultures of leukaemic or transformed cell lines. With the supernatants from these cultures they performed two types of experiments: (a) The supernatants were banded in sucrose density gradients. At the 1.16 gm/ml band (and sometimes at other band(s) – at least in Montagnier’s group’s experiments, this is not made clear), they found fragments of RNA of certain lengths (although no two had the same length) or were rich in adenine, (poly(A) rich fragments), and called these "HIV RNA", the "HIV genome". Using a (dT) primer the "HIV RNA" was transcribed into a complementary DNA (cDNA); (b) The supernatants were introduced into another set of the transformed and leukaemic cell lines as well as into stimulated cultures of normal T-cells. The DNA from these cells, as well as the DNA from the cultures to which no supernatant was added, were hybridized using probes from the cDNA. Positive results were obtained only with the DNA from the cells to which the supernatants were added. This evidence was interpreted as proving that the "HIV DNA", the retro-virus, originated from the AIDS patients and in fact that these patient acquired it from the outside, that is, the retrovirus was exogenous. There are many problems associated with these experiments and their interpretation. Among the many questions their conclusion raises the most obvious are: 1. HIV is said to be a retrovirus and retroviruses are particles which contain among other things, RNA. How then is it possible to claim that the RNA which banded at 1.16 gm/ml, "HIV RNA", is the genome of a retrovirus without proof that it is a constituent of a particle, viral or non-viral which bands at this density? 2. RT is not specific to retrovirus and in fact A(n).dT15 can be reverse transcribed by all cellular DNA polymerases a, b and g. Is it possible then to consider reverse transcription of A(n).dT15 as proof for HIV isolation or even detection of a retrovirus? Even if the process of reverse transcription were specific to retroviruses, can the detection of a process ever be considered proof for the isolation of an object, in this case, retroviral particles? 3. Cell culture supernatants will contain both DNA and RNA including some enclosed in cellular debris (fragments) especially if cellular viability is not one hundred percent as is the case in cultures used by the three groups. The RNAs may include messenger RNA (which is adenine rich), as well as high molecular weight heterogenous nucleic RNA. These RNAs, in addition to having high molecular weight and heterogeneity in size, also have poly(A), with the poly(A) attached at the 3’ end of the molecule, and may be RNAase resistant. Actinomycin inhibits its synthesis and also interferes with its proper processing and breakdown.99 From animal virology it is also known that non-retroviral RNA and DNA also bands at 1.16 gm/ml.100 How is it then possible to claim that just because an RNA bands at 1.16 gm/ml and is adenine rich or has a certain length, it is "HIV RNA"? If this RNA is "HIV RNA", then what is the other RNA and the DNA which also bands at this particular density? If the latter are cellular why not the poly(A)RNA as well? 4. By definition, retroviruses are infectious particles which contain only RNA. When they enter a cell the RNA is reverse transcribed into DNA, which is then integrated into cellular DNA as a provirus, which means that "HIV DNA" will be present only in the cell and nowhere else. Yet many HIV experts including Gallo have shown that both the supernatants of "infected" cell cultures and the "HIV particles", that is, the material which bands at 1.16 gm/ml, contains "HIV DNA" which "may integrate directly into the host chromosomal DNA". 101-103 The question then arises, is the "HIV DNA" the result of "HIV RNA" reverse transcription or is it vice versa? 5. It is accepted that the HIV RNA is localized in a condensed core surrounded by a "lipid-bilayered envelope derived from the cellular membrane of the host cell, studded with virally encoded gp120 and myristylated protein, p17. The so-called core-envelope link (CEL) attaches the core to the envelope". 103 One of the best know facts in biology is that condensed cores (chromatin) is transcriptionally inactive. This is one of the reasons why viruses, including retroviruses, to multiply, must first enter cells where their chromatin is decondensed. However, in a paper published in 1993, Hui Zhang and colleagues including Poiesz, from Suny Health Science Center at Syracuse, New York, wrote: "We have shown that in the absence of detergent, large amounts of DNAase-resistant viral DNA can be synthesized within intact HIV-1 virions, indicating that this phenomenon is not dependent on perturbation of the viral envelope. [Not to mention econdensation of chromatin]. Nascent viral DNA synthesis also occurred in purified virions incubated at 37° in cell-free human physiological fluids including seminal plasma, breast milk, and fecal fluids".103 This means that either: (i) the "intact HIV-1 virions" perform a function that no other biological system with very condensed and protected chromatin can perform; or (ii) the "HIV RNA" found in the supernatants or in the "purified virions" is present in an unembodied form; or (iii) the "HIV RNAs" are de novo synthesized in the cell cultures (see 6.3.5); 6. At present there is ample evidence that any RNA or DNA present in the supernatant, irrespective of its origin, especially when cells are stimulated by polycations and oxidizing agents, will be taken up by the cells (see 7.1). How is it then possible to claim that a positive hybridization signal in cells cultured with the same "HIV DNA" containing supernatant as the supernatant from which the "HIV DNA" probe originated but not in other cells is proof that the "HIV DNA" is the genome of an exogenous retrovirus? 7. The first, absolutely necessary step in proving that the "HIV DNA" originated from the lymphocyte cells of AIDS patients and those at risk, is to perform hybridization experiments using the DNA of their fresh, uncultured lymphocytes and the "HIV DNA" as a probe. It is hard to understand why neither Montagnier’s nor Levy’s group reported such experiments. Gallo’s group did and the results were negative (see 6.4.4). How is it then possible to claim that "HIV DNA" is the genome of an exogenous retrovirus which originated from AIDS patients and those at risk? 8. Reading the seminal paper on HIV isolation entitled "Detection, Isolation and Continuous Production of Cytopathic Retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS", one gets the impression that the leukaemic HT cell line which Gallo, Popovic, and their colleagues used was a new cell line and one which they established. The Gallo inquiry revealed that the HT (H9) cell line is the same as that used by Levy’s group, HUT78, a leukaemic cell line established in another laboratory. However, the abundant evidence for the existence of endogenous human retroviruses has largely been obtained from experiments on leukaemic and transformed cells. Evidence exists that both H9 and EBV-transformed B lymphocytes release retrovirus-like particles even when not "infected with HIV". 104 Furthermore, the HUT78 (H9) 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-I. Indeed, as far back as 1983, he claimed to have shown that the HT (H9) cell line contained HTLV proviral sequences. 105 According to some American researchers, EBV-transformed normal human peripheral blood B lymphocytes contain HTLV-I related transcripts.106 Since all retroviral particles by definition band at 1.16 gm/ml, assuming that all the groups had a retrovirus at this density, how is it possible to claim that the retrovirus originating from the HUT78 and EBV-transformed B-lymphocytes is a new retrovirus HIV, and not one which was already present? Can one claim that the "HIV RNA" and thus the probes and primers originating from it are the RNA and probes and primers of a unique exogenous retroviral genome? 9. The biological dogma states that DNA is synthesized on a DNA template, RNA on a DNA template, and proteins on an RNA template. In other words, the only way for a cell to acquire new nucleic acid entities is for them to be introduced from the outside, exogenously either from another cell type, an infectious agent or a synthetic nucleic acid. If the biological dogma is correct then the "HIV RNA", be it a cellular or viral molecular entity, should have originated either from the patients’ lymphocytes or the transformed and leukaemic cell lines. However, when "HIV cDNA" was used a probe, not one of the groups reported positive hybridization results from any of the cells, not even from the lymphocytes of AIDS patients. The question then arises, does a unique molecular entity, "HIV DNA" exist? What does it mean and from where did it originate? 6.3. SPECULATIONS ON "HIV DNA" If one wishes to speculate on the nature and origin of RNA (cDNA) derived from the cultures containing tissues of AIDS patients and those at risk, and which bands at 1.16 gm/ml, there are many possibilities including: 6.3.1 Although to date no such evidence exists, it is possible that the stretch of RNA, presently called "HIV RNA", is the genome of an exogenous retrovirus, HIV. However, for this to be considered proven in addition to satisfying all the requirements in 6.1 one must also show that: (i) the unique stretch of RNA can be obtained only from cultures of particular individuals; (ii) when the RNA (or cDNA) is used as a probe to test fresh, uncultured lymphocytes, a positive test is obtained only from the fresh cells of individuals who also have a positive culture; (iii) that in animals or humans, the retrovirus is horizontally (animal to animal, person to person) transmitted. 6.3.2 The genome of an endogenous retrovirus, that is, a stretch of RNA with a corresponding DNA template 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. For many decades it has been known that animal DNA contains sequences "closely related or identical with those of infectious viruses". However, the human genome was considered to be an exception and as late as 1994, both Gallo and Fauci were of the opinion that "...there are no known human endogenous retroviruses". 107 In fact, in the 1970s and in the 1980s after Gallo’s claim of the discovery of HL23V, HTLV-I and later HTLV-II, and especially after Montagnier’s claim of the discovery of HIV, considerably greater interest was engendered in retroviruses with the result that it became "increasingly clear that the DNA of man, like that of other vertebrates, contains many integrated retroviral genomes", 25,108 and that in many cases the genes are expressed, "including mRNA transcripts related to full-length endogenous retroviral DNA" 109,110 with open reading frames for the gag, pol and env proteins. 111 By 1987, many researchers reported the expression of the genome of the human endogenous retrovirus, HERV-K, homologous to the mouse mammary tumor virus (MMTV). "In several cell lines, HERV-K genome was expressed as an 8.8 kilobase poly(A)+ RNA which appears to be the full-length transcript of this genome". When the human breast cancer cell line T47D was "grown in RPMI 1640 supplemented with 10% fetal calf serum, HERV-K genome expression was slight". However, when the cells were treated with estradiol and then progesterone, they produced "retrovirus-like particles and soluble protein sharing antigenic determinants with MMTV env gene product". 112 In support of their thesis "that a human endogenous RT might mediate gene movements leading to leukemia and cancer", researchers from Hahnemann University, Philadelphia, including David Gillespie, a long time collaborator of Gallo "demonstrated the presence of a reverse transcriptase-like enzyme in retroviral particles from patients with essential thrombocythemia, polycythemia vera, and chronic myelogenous leukaemia. It was subsequently shown that the human genome contains 50 copies of HERV-K. HERV-K is a human endogenous class I retroviral element that contains gag, pol and env open reading frames...as well as intact LTR regions...Expression of a 9 kb genomic HERV-K RNA transcript was detected in human cell lines...We were able to show for the first time the expression of HERV-K pol gene in human blood leukocytes. The HERV-K pol gene was expressed in peripheral blood cells from two sets of non-leukemic individuals. The first set consisted of seven normal donors, while the second set consisted of 3 patients with PV, all of which expressed HERV-K pol gene. Five different nucleotide sequences were obtained from the seven normal donors. Four of the five normal sequences contained heterogenous open reading frames for pol as detected by both RT-PCR and RNAase protection. Unlike normal donors which randomly express HERV-K proviruses, analysis of HERV-K pol from PV patient showed selective expression of a restricted family of related proviruses". 113 By 1995, Gallo admitted that the human cell does contain retroviral genomes but he still insisted they are defective, "Retroviruses are transmitted either genetically (endogenous forms) or as infectious agents (exogenous forms). As do many other animal species, humans have both forms...The DNA of many species, including humans, harbor multiple copies of different retroviral proviruses. The human endogenous proviral sequences are virtually all defective, and comprise about one percent of the human genome". 114 The view regarding defectiveness is not shared even by Reinhard Kurth who, with his colleagues, has extensively studied the human endogenous retroviruses115 and have shown that HERV-K sequences are transcribed and that a human teratocarcinoma cell line, GH, which contains these sequences, when examined by EM was found to produce "human teratocarcinoma-derived retrovirus (HTDV) particles". By 1993 Kurth and colleagues reported that in the GH cell line, "Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic RNAs are generated by single or double splicing events...Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family". Discussing their findings they wrote: "In Northern blots, expression of HERV-K could only be demonstrated in teratocarcinoma cell lines but not in other human lines. Preliminary RT PCR studies suggest, however, that HERV-K may be expressed in many if not all human cells at levels too low to be detectable in Northern blots. The basis of the significant quantitative differences in expression between teratocarcinoma cells and other cell lines is not clear. It is intriguing to speculate that a cellular factor(s) may regulate the synthesis of HERV-K mRNA depending on the cell type or the state of differentiation. In this context, it should be remembered that other retroid elements [ERV-9, RTLVL-H, LINE-1] are also preferentially expressed in human teratocarcinoma cells".116 It is of interest to note that Montagnier and his colleagues reported their "HIV genome" from a transformed cell line, that Levy and colleagues’ HUT78 cell line is a human leukaemic cell line and that Gallo and colleagues’ H9 cell line is none other than HUT78, and thus must have HTLV-I as well as endogenous retrovirus. It is equally important to note that although Kurth et al found no sequence homology between HERV-K and "human T-lymphotropic virus" or HIV, many researchers reported HTLV-I sequences in the human genome including in cell lines derived from teratocarcinoma. In a paper published in 1985 researchers from a number of institutions in the USA including the Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, reported that "Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D virus. The HLM-2 gag and pol genes share extensive homology with those of the M432 retrovirus (a type A-related retro-virus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70% identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of the M432 viral genome". The pol region of HLM-2 showed homology with HTLV-I which, according to the authors "is not endogenous to human cells but is transmitted horizontally as an infectious tumor-inducing virus of humans". 117 In 1987 researchers from Canada reported the finding of a "Human Endogenous Retrovirus-like Genome with Type C pol sequences and gag sequences related to the Human T-cell Lymphotropic Viruses", HTLV-I and HTLV-II. 118 In 1989 researchers from the Department of Biochemistry, New York University showed that "human DNA contains a wide spectrum of retrovirus-related reverse transcriptase coding sequences, including some that are clearly related to human T-cell leukaemia virus type I and II, some that are related to the L-I family of long interspersed nucleotide sequences, and others that are related to previously described human endogenous proviral DNAs. In addition, human T-cell leukaemia virus type I-related sequences appear to be transcribed in both normal human T cells and in a cell line derived from a human teratocarcinoma". 119 In a paper published in 1989, researchers from the USA summarized their experimental findings as follows: "Human T-cell leukemia virus (HTLV) type I-related endogenous sequences (HRES) have been cloned from a human genomic library. HRES-1/1 is present in DNA of all normal donors examined. By nucleotide sequence analysis, HRES-1/1 contains two potential open reading frames capable of encoding a p25 and a p15. A 684 flanking region 5’ from the first ATG codon of p25 contains a TATA-box, a polyadenylation signal, a putative tRNA primer binding site, and inverted repeats at locations which are typical of a retroviral long terminal repeat...The HRES-1/1 genomic locus is transcriptionally active in lymphoid cells", including EBV-transformed normal human peripheral blood lymphocytes, leukemic cell lines, melanoma cells and embryonic tissues. 106 In a paper published in 1992 by researchers from Hungary and Britain entitled "Human T-cell lymphotropic virus (HTLV)-related endogenous sequences, HRES-1, encodes a 28-kDa protein: A possible autoantigen for HTLV-I gag-reactive autoantibodies", the "presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, in the human genome was documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames...Antibodies to HRES-1-specific synthetic peptides were noted in patients with MS, progressive systemic sclerosis (PSS), SLW, Sjogren syndrome (SJS), and essential cryoglobulinemia (ECG). The data suggest that HRES-1 may serve as an autoantigen and correspond to a natural target of HTLV-I core protein-reactive autoantibodies". 120 6.3.3 The genome of a retrovirus de novo assembled by genetic recombination and deletion of: (a) endogenous retroviral sequences; (b) retroviral and cellular sequences; (c) non-retroviral cellular genes. In the virological literature there is ample evidence which shows that when a cell contains two proviruses, progeny may be found that possess the genome of one but the structural proteins of either or both viruses present. Conversely, the RNA may be viral but at least some of the proteins may be cellular. In other instances, the particles do not have a genome at all, or one or more genes are missing (genetically defective viruses). The genetic mixing can be between viral genomes or between viral and cellular genes. 83,121 According to distinguished retrovirologists such as Weiss and Temin, new retroviral genomes may arise by rearrangement of cellular DNA caused by many factors including pathogenic processes, a view that proposes retroviruses as an effect and not the cause of disease. 122,123 According to Varmus, "Retroviral genomes recombine at high frequency (estimates range as high as 10 to 30% for each cycle of multiplication), and heterodimeric RNAs are thought to be intermediates, with recombination taking place during reverse transcription. Recombination appears to be strongly favored by homology, but joining also occurs occasionally between unrelated sequences, e.g., during the latter phase of genetic transduction by retroviruses. When viruses are grown in cells that contain related endogenous proviruses, packageable transcripts from those proviruses may participate in recombination reactions with the exogenous virus. This is most dramatically revealed by the repair of deletion mutations in the genome of an exogenous virus in a fashion that superficially resembles gene conversion". In some animals proviruses have been acquired "during recent breeding of the strains in the laboratory" and "in a few instances, endogenous proviruses have been established or increased in number during experimental observations" 121 (italics ours). As far back as 1974, based on the then available evidence, Howard Temin proposed that the retroviral (ribodeoxyviruses) genomes originate from "normal cellular components. The relationships between the different ribodeoxyvirus groups reflect the relation-ships among the cellular components from which the viruses evolved and the convergent evolution of the viruses. In other words, there are relationships among ribodeoxyviruses because the ribodeoxyviruses evolved from cells which themselves had relationships deriving from common ancestors. A possible mechanism of this evolution is described in Fig. 5". In the legend to Fig. 5 Temin wrote. "A section of a cell genome becomes modified in successive DNA (W) to RNA (-) to DNA transfers until it becomes a ribodeoxyvirus genome. First, these sequences evolve as part of a cellular genome. After they have escaped as a virus, they evolve independently as a virus genome. The time scale may be millions of years in germ-line cells and days in somatic cells". 122 Temin reinforced his view in a more recent publication. 124 In 1975, Gallo, Gillespie and their colleagues wrote: "Even though RNA of class II [exogenous] retroviruses shows minimal homology to uninfected host cell DNA, hybridization of nucleic acids among class II leukemia viruses from different species gives a pattern which is the same as the phylogenetic relatedness among their natural hosts...We have proposed that these and other results favor the interpretation that all RNA tumor viruses are derived from cell genes, a proposal in agreement with the virogene theory...By analysis of the RNA of viruses infecting and replicating in a new host, evidence has also been obtained which indicates that the genome of type C viruses can be substantially changed by the host, probably by recombination with host DNA". 125 A few years later, Coffin wrote: "The close relationship of virion proteins as well as overall nucleic acid homology must mean that both exogenous and endogenous avian tumor viruses [retroviruses] derive from a common ancestor". 126 In 1991 researchers from the New York University published a paper entitled, "Evolutionary Implications of Primate Endogenous Retrovirus". Discussing the presently available data they wrote, "A recent detailed phylogenetic analysis of exogenous and endogenous retroviruses (including retrotransposons) strongly suggests that a pool of endogenous retroviral sequences periodically contributes to the generation of exogenous viruses, and that the presence of endogenous primate retroviruses is probably more directly related to exogenous viruses that might have been thought"; 127 6.3.4 The "novel" RNA found in the cell culture supernatant and the material from it banding at 1.16 gm/ml, the "HIV RNA", may have nothing to do with a retroviral genome. It may be 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. 128,129 Not only can retroposition "shape and reshape the eukaryocytic genome in many different ways" 128 but the nonviral retroelements may be similar to the retroviral elements. According to Doolittle et al from the University of California, San Diego,"...the entire group of reverse transcriptase-bearing agents, including retrotransposons and genuine retroviruses, has recently been dubbed, "retroids". Sequence comparisons by many other workers leave little doubt that the reverse transcriptases of all the "retroids" considered here are homologous, which is to say, the sequence resemblances are not the result of chance or convergences. Our own comparisons confirm that general notion, not only for reverse transcriptases, but also for the ribonucleases, endonucleases and proteases, although it should be under-stood that not all "retroids" contain all four enzymes...All of these elements have additional features in common with retroviruses including characteristic LTRs (long terminal repeats) and primer sites that are complementary to various tRNAs. Like retroviruses, most contain distinctive nucleic acid-binding and core particle proteins; in electron micrographs there is a remarkable likeness to retroviral capsids...About the only feature that regularly distinguishes many of these retrotransposons from genuine retroviruses is the absence of an envelope protein". 17 6.3.5 Although 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, at present it is still generally accepted that any time one finds a particular stretch of RNA in a cell, for example, in a T-lymphocyte, unless RNA or DNA has been introduced from outside, all T-cells, regardless of their physiological state or stresses to which they are subject, will contain a corresponding stretch of DNA. In other words, the DNA (genes) in a cell are invariant and all RNA molecules in the cell are subservient to a matching length of DNA. However, according to McClintock, the genome can be restructured and not only by transposition. In her Nobel lecture of 8th December 1983, she said, "rapid reorganization 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" 130 [italics ours and see this reference for examples]. In the 1980s a number of phenomena have been discovered which brought startling changes in concepts including the following: Up until the late 1970s, the prevailing concept was that a discrete, contiguous stretch of DNA is a structural gene encoding the genetic information to specify the manufacture of a single protein, and that the linear sequence of the nucleotides in this stretch of DNA corresponds directly to the linear sequences of the RNA nucleotides and to the amino acids in the protein. The first discovery which contradicted this belief was the discovery that the DNA base sequences which coded for a given protein were not in a continuous stretch of DNA but may be interspersed with other, non-coding base sequences, that is, the genes are split, "genes-in-pieces". A number of mechanisms have been postulated to account for this observation. In one such explanation it is hypothesized that the entire stretch of DNA is transcribed into a piece of RNA, then the non-coding regions (introns) are excised and the coding regions (exons) are spliced together to make the appropriate messenger RNA.131 There are no rules setting an upper limit on the number of introns in a "gene", some genes may have up to sixteen or more introns. Nor are there any rules regarding the length of introns, although in general, introns are much longer than exons, the length of exons "peaking at about 40 or 50 amino acids...the shortest intron being 50 bases long, the longest extending out to some 50.000 bp".132 According to Gilbert introns represent "hot spots" for recombination and new genes can be created "through the coupling of exons by intron-mediated recombination", "introns are lost and more complicated exons are formed". 133 At present evidence exists showing that at least some introns are mobile genetic elements, transposable elements, they self-splice, they often contain reading frames capable of encoding a protein including "regions of homology to reverse transcriptase scattered over a roughly 250-amino acid stretch in the middle of each intron ORF". 134 The discovery of split genes "shows that the genetic apparatus of the cell is more complex, more dynamic than any of us had suspected". 132 Another strongly held view was the belief that all cellular reactions and thus gene splicing were catalyzed by a protein enzyme. In the early 1980s it was found that RNA can cut, splice and assemble itself, as well as assemble RNAs other than itself. 135-138 6.3.6 One of the strongest held views in biology is the belief that nucleic acids have an inherent ability of instructing their own synthesis and that nucleic acids cannot be synthesized in the absence of a nucleic acid template. Manfred Eigen and his colleagues in Germany conducted extensive theoretical and experimental work on molecular self-replication. 139 In their experimental work they used the bacterial virus (phage) Q b . In addition to its genome, a simple strand RNA molecule of 4500 nucleotides, the virus has an RNA molecule of 220 nucleotides known as "Spiegelman’s minivariant" which, like the genomic RNA, is reproduced in cell-free laboratory systems by an enzyme called Q b replicase. By mixing Mg 2+ ions, the nucleoside triphosphates ATP, GTP, UTP, CTP, Q b replicase and template RNA, they could obtain RNA replication but a totally unsuspected finding was that even the absence of the template, RNA was still synthesized. They performed many experiments to prove this phenomenon and to exclude the possibility of the presence of an initial RNA tem-plate and concluded, "Finally we were convinced we had before us RNA molecules that had been synthesized de novo by the Q b replicase enzyme. What was most puzzling, the de novo product had a uniform composition which in each trial turned out to be similar to or even identical with Spiegelman’s minivariant". When the template free mixture was then divided into several isolated compartments where optimal conditions for de novo synthesis were maintained they found that "each component had a uniform population of de novo product, the products differed from compartment to compartment. Further analysis revealed however that the different sequences were not completely unrelated...There was a definite, uniform final product for any set of experimental conditions, but here were as many different optimal products as there were different experimental conditions. One of the optimal products appeared to be Spiegelman’s minivariant... Other products of optimization were adapted to conditions that would destroy RNAs, such as high concentrations of ribonuclease, an enzyme that cleaves RNA into pieces...Some variants were so well adapted to odd environments that they had a replication efficiency as much as 1000 times that of variants adapted to a normal environment... Any RNA formed by noninstructed chemistry would be reproduced by template-instructed chemistry at a rate proportional to the current RNA concentration. The result would be exponential growth. Furthermore, even if only a single template were formed initially by noninstructed synthesis, there would soon be a host of different sequences because errors (point mutations, insertions and deletions) would inevitably be made in the course of replication. Hence in each generation there would be not only a larger number of RNA strands but also a greater variety of RNA sequences. What would happen then? Some of the mutants would be copied more rapidly than others or would be less susceptible to errors in copying, and their concentration would increase more rapidly. Sooner or later these faster-growing mutants would take over...Hence the results of the self-replication competition had to be the master sequence together with a huge swarm of mutants derived from it and from which it had no way of escape...We call this entire mutant distribution a quasispecies. It is the quasispecies mutant distribution that survives the competition among self-replicating RNAs and not just one master sequence or several equivalent ones that are the fittest genes in the distribution. The essence of selection then is the stability of the quasi-species". 140 According to Eigen and his colleagues, the maximum length of an RNA master sequence is of the order of 10,000 nucleotides. 139,141 6.3.7 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 retailored, 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!". 142-144 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 preeminence 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". 145 6.3.8 CONCLUSION The finding of a novel stretch of RNA or DNA and proteins in: (a) lymphocytes of sick individuals or individuals who have been "shocked" with agents such as physical or chemical mitogens, carcinogens or oxidizing agents in general as is the case with AIDS patients and those at risk; 77,79,90 (b) lymphocytes in cultures or co-cultures (which could lead to the appearance of hybrids) which have been additionally "shocked" with sometimes multiple, similar agents; is not proof that the given stretch of RNA comes from the outside, irrespective of its length, the presence of poly(A) and number of ORF ("genes"). From Montagnier’s, Gallo’s and Levy’s and their colleagues’ evidence it is not possible to conclude that the "HIV RNAs" they found are a "new species" of RNAs induced by "shocking" the cells or by one or more of the other phenomena which have come to light in the 1980s. Nor is it possible to conclude that their RNAs are the genome of an exogenous retrovirus as they did. However, a number of predictions can be made: (a) If the "HIV DNA" is indeed the genome of an exogenous retro-virus then:
(b) If the selected RNA which was found to band at 1.16 gm/ml, the "HIV RNA", is the genome of a retrovirus which exists "in all of us", endogenous retrovirus, then again evidence must prove the existence of a unique molecular entity, "HIV RNA", ("HIV DNA"). When hybridization studies are conducted using the full length of the unique molecular entity as a probe, positive results should be found "in all of us"; (c) If the RNA found by the three groups, "HIV RNA", is the genome of a retrovirus assembled de novo from DNA already existing in the cells, as the result of in vivo or in vitro conditions, evidence must also prove the existence of a unique molecular entity. When the whole length of the unique fragment of nucleic acids is used as a hybridization probe, a positive result should only be found in cells which are subjected to exactly the same in vivo or in vitro conditions as those from which the "HIV RNA" at 1.16 gm/ml was obtained. When only fragments of "HIV RNA" are used for hybridization, the probability of finding a positive result will increase; (d) If the "HIV RNA" is a unique non-viral molecular species of RNA resulting from the transcription of a unique molecular species of DNA then when the whole fragment of "HIV RNA", ("HIV cDNA") is used a probe for hybridization studies, a positive result should be found only in the cells of the same type as those from which the "HIV RNA" originated, in all individuals; (e) If the "HIV RNA" is neither the genome of a retrovirus nor a faithful transcript of a fragment of DNA present in the cells from which it has been obtained, but is the result of the "shock" to which the cells have been exposed, either in vivo or in vitro or both, or as a result of the phenomena discovered in the 1980s then:
6.4. EVIDENCE THAT THE "HIV RNA" BELONGS TO AN EXOGENOUS RETROVIRUS The Montagnier, Gallo and Levy groups claimed that the special RNA which they selected from the total RNA which in sucrose density gradients banded at the density of 1.16 gm/ml was novel to the lymphocytes and that in fact belonged to an exogenous retrovirus. Although they did not present evidence to prove this assertion, the possibility cannot be excluded that indeed this may have been the case. Since at present their claim is generally accepted one would have thought that by now they or other researchers should have been able to pro-vide ample confirmatory proof. This does not seem to be the case: 6.4.1 If the RNA originates from a retrovirus either endogenous or exogenous then evidence must exist which proves that such RNA is a constituent of particles which possess at least the most basic morphological and physical features of retroviruses, that is, "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)". 82 To date not only has nobody shown that the "HIV RNA" belongs to such particles, there is no evidence that particles of any kind are present in the material from cell cultures/cocultures which bands at the retroviral density of 1.16 gm/ml and from which the "HIV RNA" is selected. Furthermore, although particles have been demonstrated in cultures, cultures contain many different types of particles but none display BOTH principal morphological characteristics, that is, "a diameter of 100-120 nm" AND surfaces which "are studded with projections (spikes, knobs)". 146 6.4.2 If the "HIV RNA" is the genome of an exogenous retrovirus then, like the "exogenous animal retroviruses", one should be able to find it in infected material without the necessity to revert to the use of co-cultivation or mitogenically stimulated cultures. However, none of the phenomena which are thought to prove the existence of HIV can be detected unless one employs mitogens or co-cultures or both (and sometimes additional "shock"), a fact accepted by both Montagnier and Gallo. 78,147 6.4.3 One cannot claim that "HIV RNA" is the genome of a unique retrovirus, HIV, unless evidence is presented to prove that ‘HIV" is a unique molecular entity. By 1985 it was known that "the env genes of ARV and HTLV-III differ by more than 20 percent" and that "the Gallo group has sequenced another HTLV-III isolate and finds that it differs from the first by about as much as ARC". 114,148 By 1986, Gallo and his colleagues accepted that the "HIV genome" has a "far greater variability" as "compared to HTLV" and in fact "The rate of genetic change for the AIDS virus is more than a million fold greater than for most DNA genomes and may even be tenfold greater than for some other RNA viruses including certain retroviruses and influenza A virus". 149 At present it is accepted that "no two isolates are identical. Each isolate contains many variants". 150 In one and the same patient the genomic data in monocytes differs from that in T-lymphocytes. 151 There are "striking differences" between the proviral DNA and cDNA in one and the same PBMC sample "which could not be explained by either an artefact of reverse transcriptase efficiency or template selection bias". 152 The genetic data obtained in vitro do not correlate with the data obtained in vivo, "to culture is to disturb".153 According to the researchers from the Pasteur Institute "an asymptomatic patient can harbor at least 106 genetically distinct variants of HIV, and for an AIDS patient the figure is more than 108. 154,155 The "HIV genome" varies with time; in one case where clones were obtained 16 months apart all the clones detected in the second sample were distinct from the clones in the first sample. 156 It is also accepted that up to 99.9% of the "HIV genomes" may be defective. 157 According to Levy, "The mechanism responsible for generating these varying strains of virions is puzzling. One theoretical possibility is that the unintegrated proviral copies of HIV that accumulate during acute replicative infection can undergo efficient genomic recombination leading to the evolution of infectious variants." 158 In Robin Weiss’ view, "the source of variation is the infidelity of reverse transcription, which has no editing mechanism for transcriptional errors", as well as "genetic recombination" especially when cell fusion takes place. 159 By the late 1980s, researchers from the Pasteur Institute concluded, "it is increasingly clear that it will be very difficult to describe correctly the characteristics of HIV viruses using single molecular clones". "It is evident that HIV, either in vivo or in vitro, is extraordinarily complex and that a population-based approach", a quasi-species approach as defined by Eigen, must be used to describe HIV. They also added, "Even with a population-based approach, only small regions of the HIV genome can be studied... Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult". 153,160 The data which have been published since confirm their conclusion. Prior to the 1990s, the HIV sequences were classified as African and USA/European with sequence differences of 20- 30 percent between these two groups.161 In the 1990s, HIV researchers started to divide the "HIV genome" into subtypes A, B, C, D, E, etc. The basis for this classification system is: "(a) subtypes are approximately equidistant from one another in env (a ‘star’ phylogeny); (b) the env phylogenetic tree is for the most part congruent with gag phylogenetic trees; (c) two or more samples are required to define a sequence sub-type". However, "Subtype naming problems have arisen for several reasons. A small but not insignificant number of viral sequences are hybrid, clustering with one sequence subtype in gag and another sequence subtype in env, for example; or, to take another example, clustering over different stretches with two or more subtypes in env...Naming becomes problematic when highly divergent forms of a given subtype arise: such forms are sometimes designated A’, B’, F’, etc. It is increasingly necessary to have sequence data from both gag and env coding sequences when a new form or subtype is being claimed". 162 By the middle of this year "at least ten" (A-J) prevalent major (M) and a low prevalence, O, HIV-1 genotypes were described and new genotypes are still reported. 8,163. According to researchers from the Henry M Jackson Foundation Research Laboratory and Division of Retrovirology, Walter Reed Army Institute, USA, "The great majority of genotypic consignments for HIV-1 are based on subgenomic genome", and not by comparisons of the whole genome. This is because, "it remains impractical to obtain full length genomic sequences of HIV-1 isolates as a routine genotyping method, due to the low abundance of HIV-1 proviral DNA in clinical samples and virus cultures on PBMC substrate, and to the relative inefficiency of the polymerase chain reaction when amplicons become large". "The designation Human Immunodeficiency Virus Type-1 (HIV-1) encompassed an unanticipated complexity of viral forms".163 . According to researchers from the Los Alamos National Laboratory, "while a sub-type designation based on a gene or gene fragment may be correct, recombination may have occurred. Therefore, care should be taken to not over interpret the subtype designation. If one is to discuss the subtype designation of viral isolates based on the data presented here, they should refer to the designation as ‘B-like over V3 loop region’ rather than as ‘subtype-B’". 164 One and the same person may be "infected" with more than one subtype. 165 This means that at present it is not possible to say what are the sequence differences, both qualitative and quantitative, between different HIV-1 subtypes. Nonetheless, some suggestive data does exist. In 1993 researchers from several institutions "reported that in the A-G HIV-1 genotypes the intra-genotypic gag distances averaged 7% whereas the inter-genotypic distances averaged 14%...The maximum level of variability in gag is still well below that observed for the envelope region of HIV-1". 166 "Two HIV-1 strains, designated ANT70 and MVP5180 were isolated in 1987 and 1991 respectively from patients in Cameroon". They were classified as HIV-1 subtype O. By 1994 evidence was presented which "indicated that subtype O was endemic in Cameroon and Gabon". 167 "DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49% respectively...Comparison of the MVP-5180 amino acid sequence with that of the Gabon chimpanzee virus showed similarities of 70, 78 and 53% in the gag, pol, and env genes, respectively; similarities of 70, 76 and 51% to the Uganda HIV-1 (U455) and of 54, 57 and 34% to the HIV-2 isolate D205 were found". The researchers from Germany and Cameroon who conducted this study expressed the view that "Even more divergent HIVs may exist. Such divergent HIVs are likely to be transmitted by the usual routes (sexual and blood contact and mother-to-infant transmission), leading to wider distribution. They will have to be taken into account in vaccine development and diagnostic test sensitivity and specificity". 168 Indeed, this seems to be the case. Last year, David Ho and his associates169 studied an Australian patient with "primary infection". "Since seroconverters generally harbor a relatively homogenous population of viruses", they were surprised when they found that he was "co-infected", "by multiple subtype B HIV-1...The average genetic distances between group I and II, I and III, and II and III were 9.6, 16.5 and 18.4% respectively... One population of sequences was clearly distinguishable from the others on the basic of phylogenetic analysis. In addition, sequences suggesting recombination between two of the three distinct viral populations were also found". That the "HIV DNA" may be "Even more divergent" than has been generally accepted is best illustrated in a study published this year by researchers from the United States. Because protease inhibitors are becoming the drugs of choice for the treatment of "HIV infected" individuals, and because "naturally occurring mutations in HIV-1 infected patients have important implications for therapy and the outcome of clinical studies", these researchers performed a "sequence analysis of the pr gene [protease gene] in 167 HIV-1 viral strains from 102 pro-tease inhibitor naive patients collected from different geographic regions of the United States". "Given the enzyme’s relative small size and the constraints on its structure imposed by function, it was reasonable to conclude that sequence variability in HIV-1 would be limited". To their surprise it was found that "A total of 41% of the nucleotides and 49.5% (49/99) of the amino acids were variable. The amino acid diversity seen in these USA viral isolates is much greater than that previously reported for HIV-1 clade B viruses" and is also greater than that seen in pr genes for all HIV-1 clades (40 out of 99, 40% of amino acids varying)"! 170 At present, more so than in 1986 when Gallo and colleagues reached their conclusion that "The rate of genetic changes for the AIDS virus is more than a million fold greater than for most DNA genomes and may even be tenfold greater than for some other RNA viruses including certain retroviruses and influenza A virus", and in 1989, when the Pasteur researchers reached their conclusion that "the task of defining HIV infection in molecular terms will be difficult", there is no evidence which proves the existence of a unique molecular entity "HIV RNA" ("HIV DNA"). In fact, there are a number of reasons why the myriads of incommensurable "HIV DNAs" cannot be even described "in terms of populations of closely related genomes, referred to as a quasispecies". 153 These include: (a) Eigen and his colleagues developed the quasispecies model to describe the distribution of self-replicating RNAs. However, the "HIV RNA", is said not to be a self replicating RNA, but replicates through a DNA intermediate; (b) the self-replicating RNA of the RNA viruses appears to "demonstrate remarkable stability in some situations. The type 3 Sabin poliovirus vaccine differed from its neurovirulent progenitor at only 10 nucleotide positions after 53 in vitro and 21 in vivo passages in monkey tissues. In 1977, H1N1 influenza A virus reappeared in the human population after 27 years of dormancy with sequences mainly identical to those of the 1950s virus". Although Eigen’s quasispecies model has been used to describe the genome of RNA viruses, even 1% sequence differences in these genomes are considered to represent "extreme variability". "Many selective forces may stabilize virus populations. These stabilizing factors may include the need for conservation of protein structure and function, RNA secondary structure, glycosylation sites, and phosphorylation sites. Even third-codon changes can be subject to selective pressures. Recently, remarkable conservation of certain protein domain sequences has been observed between completely unrelated RNA viruses. 171 Is it possible then to describe the "HIV DNA" even if it has variation of 10% , not to mention 20 or 30 or 40% as is the case, as a "population of closely related genomes, referred to as a quasispecies"?; (c) Defining the concept of a quasispecies Eigen wrote: "In the steady state that is eventually reached the best competitor, designated the master sequence m, coexists with all mutant sequences derived from it by erroneous copying. We designate this distribution of sequences as quasispecies". However, to date, nobody has proven that:
6.4.4 If the "HIV RNA" stretch is the genome of an exogenous virus which infects individuals with AIDS or those at risk, then this RNA (or cDNA) should be present in fresh uncultured tissue from all these individuals and in nobody else. Furthermore, if in these individuals there is massive HIV infection, as some of the best known HIV experts claim, 171,173 Southern blot hybridization should be more than sufficient to detect it. The first such study was conducted by Gallo and his colleagues in 1984. Using a Southern blot hybridization technique they tested many tissues from AIDS patients, including lymph nodes. Summarizing their finding they wrote, "We have previously been able to isolate HTLV-III from peripheral blood or lymph node tissue from most patients with AIDS or ARC" (they "isolated" it from approximately 50% of patients referred to by Gallo). "However, as shown herein, HTLV-III DNA is usually not detected by standard Southern blotting hybridization of these same tissues and, when it is, the bands are often faint...the lymph node enlargement commonly found in ARC and AIDS patients cannot be due directly to the proliferation of HTLV-III-infected cells...the absence of detectable HTLV-III sequences in Kaposi’s sarcoma tissue of AIDS patients suggests that this tumor is not directly induced by infection of each tumor cell with HTLV-III...the observation that HTLV-III sequences are found rarely, if at all, in peripheral blood mononuclear cells, bone marrow, and spleen provides the first direct evidence that these tissues are not heavily or widely infected with HTLV-III in either AIDS or ARC". 96 These studies were confirmed by many other researchers. The finding that when the results were positive the hybridization bands were "faint", "low signal" was interpreted as proof that HIV seropositive individuals contain HIV DNA in small numbers of cells and at low copy numbers, an interpretation which became generally accepted, although Gallo and his colleagues had an alternative explanation: "Theoretically, this low signal intensity could also be explained by the presence of virus distantly homologous to HTLV-III in these cells". 96 This alternative explanation has been ignored by everybody, including Gallo. However, at a 1994 meeting held in Washington sponsored by the US National Institute of Drug Abuse, Gallo admitted "We have never found HIV DNA in the tumor cells of KS...In fact we have never found HIV DNA in T-cells".174 Data which has come to light since 1984 suggest that Gallo’s and his colleagues’ alternative explanation may be a fact: (a) at present there is ample evidence showing that normal human DNA contains sequences related to HTLV-I and HTLV-II (see 6.3.2); (b) apparently, up until 1993, Gallo was unaware of the existence of endogenous human retroviruses, 107 which means that by "virus distantly homologous to HTLV-III" they could have meant none other than the exogenous retroviruses Gallo claimed to have discovered earlier, that is, HTLV-I and HTLV-II. However, at present even Gallo admits that the human endogenous proviral sequences "comprise about one percent of the human genome"; (c) some of the best known HIV experts including Montagnier, Blattner and Gelderblom agree that the pol and gag genes "may be highly conserved between subtypes of virus" (see 5.6). In a paper published in 1996 by Reinhart Kurth and his colleagues one reads, "Retrotransposons evolved in a variety of organisms ranging from protozoa to human beings. In these elements, RT genes are linked to genes that code for polyproteins with the potential to self aggregate and to form core particles. These proteins are the equivalents of the retroviral capsid proteins usually designated group-specific antigens (gag)...They [retrotransposons] may be either the derivative or predecessors of retroviruses. Retroviruses differ from retrotransposons by the presence of at least one additional coding region, the envelope (env) gene". 175 In 1984, Gallo’s group reported that the "HIV genome" hybridized with the "structural genes (gag, pol, and env) of both HTLV-I and HTLV-II.56 Obviously, the finding of a positive hybridization "signal" at least with an "HIV" gag or pol probe is no proof for the existence of the "HIV genome"; In fact, at present evidence also exists which shows the presence of "HIV" sequences in non-infected tissues: (i) although it is no longer accepted that HIV is transmitted by or is present in insects, in 1986 researchers from the Pasteur Institute found HIV DNA sequences in tsetse flies, black beetles and ant lions from Zaire and the Central African Republic; 176 (ii) in 1985 Weiss and his colleagues reported the isolation, from the mitogenically stimulated T-cell cultures of two patients with common variable hypogammaglobulinaemia, a retrovirus which "was clearly related to HTLV-III/LAV" Evidence included positive WB with AIDS sera and hybridization with HIV probes; 177 (iii) DNA extracted from thyroid glands from patients with Grave’s disease hybridizes with "the entire gag p24 coding region" of HIV; 178 (iv) In a study designed to address the question whether the neuronal cells of patients with AIDS dementia complex are infected with HIV, "the brains from 10 patients with AIDS and neurological evidence of viral encephalitis and the brains from five patients without HIV-1 infection" were examined using an HIV gag probe. "The anti-sense riboprobe hybridized to cells known to be infected with HIV-1. It hybridized to HIV-1-infected A3.O1 cells as well as splenic and renal lymphocytes obtained at autopsies from patients known to have AIDS. The probe did not, however, hybridize to neurones in the brain sections from 10 patients with AIDS...Surprisingly, when we applied the control sense HIV-1 gag probe to the brain sections from patients with AIDS, we observed specific hybridization to neuronal cells. Similarly, when brain sections from five individuals not infected with HIV-1 were examined, the HIV-1 sense probe detected transcripts in neuronal cells. Our Northern blot analysis confirmed these results and demonstrated the presence of a 9.0-kb polyadenylated transcript in brain tissues".179 Thus, either the positive hybridization signals obtained with the antisense probe are non-HIV-specific or, as the authors concluded, there is a neurone-specific 9.0-kb transcript that shows extensive homology with antisense gag HIV-1 sequences and this transcript is expressed in neuronal cells of both HIV-1-infected and noninfected individuals; (v) Horowitz et al, "describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee and Rhesus monkey DNAs from normal uninfected individuals". They have "demonstrated the presence of a complex family of HIV-1-related sequences" in the above species, and concluded that "Further analysis of members of this family will help determine whether such endogenous sequences contributed to the evolution of HIV-1 via recombination events or whether these elements either directly or through protein products, influence HIV pathogenesis". 180 The inescapable conclusion therefore is that the hybridization studies do not prove that T-cells or any other cells of AIDS patients and those at risk contain a unique molecular entity "HIV DNA". 6.4.5 In the second half of the 1980s, in order to rescue the concept of an "HIV genome", the HIV experts made extensive use of a newly discovered process known as the polymerase chain reaction (PCR). Although the PCR is a very useful tool in molecular biology there are many problems associated with its use in studying the "HIV genome": (a) The PCR is an extremely sensitive technique. Writing of his Nobel prize winning discovery, Kary Mullis, himself rather ironically sceptical of the HIV/AIDS hypothesis wrote, "Beginning with a single molecule PCR can generate 100 billion similar molecules in an afternoon". 181 With such amplification it is not difficult to detect even very low levels of the "HIV genome". However, "a striking feature of the results obtained" by 1990 with PCR as with the standard Southern/Northern hybridization, was "the scarcity or apparent absence of viral DNA in a proportion of patients".182 In a further effort to rescue the "HIV genome", in the 1990s researchers from the Department of Genetics, University of Edinburgh, introduced a modified version of PCR, the double PCR method or nested PCR. "The double PCR overcomes the problem of limited amplification of rare template sequences". They reported that, "Using a double polymerase chain reaction which allows the detection of a single molecule of provirus and a method of quantifying the provirus molecules, we have measured provirus frequencies in infected individuals down to a level of one molecule per 10 5 PBMCs...As a general rule, only a small proportion of PBMC contain provirus (median value of samples from 12 patients one per 8,000 cells)"...samples from 7 of our 12 patients (60%) contained one or more provirus per 104 cells...while samples from all (100%) of our patients contained one or more provirus per 80,000 cells". They concluded, "The most striking feature of the results is the extremely low level of HIV provirus present in the circulating PBMC in most cases".182 There is no doubt that PCR can "amplify a DNA-needle into a DNA-haystack" but even PCR cannot perform miracles. In a review of Neville Hodgkinson’s book, AIDS, The Failure of Contemporary Science: How a Virus That Never Was Deceived the World,183, Sir John Maddox wrote, "the virus that never was has been made more tangible" early in 1995 when "it became apparent that even in the earliest stages of infection by HIV, the virus is far from dormant". 184 Maddox is referring to two papers published in Nature in 1995. One by Ho et al where the authors claim to have shown that in patients who have not received antiviral treatment the "plasma viral levels ranged from...15 X 10 3 to 554 X 10 3 virions per ml"; 172 the other by Wei et al where it is claimed that the "plasma viral RNA levels in the 22 subjects at baseline ranged from 10 4.6 to 10 7.2 molecules per ml" and concluded that their study "suggests that virus expression per se is directly involved in CD4 + cell destruction. The data do not suggest an "innocent" bystander mechanism of cell killing whereby uninfected or latently infected cells are indirectly targeted for destruction by absorption of viral proteins or by autoimmune reactivities". 173 These claims raise two obvious questions: (i) "The majority of exogenous pyrogens are microorganisms, their products or toxins", and "endogenous pyrogens are polypeptides produced by a large variety of nucleated host cells including monocyte/ macrophages" and "lymphocytes, endothelial cells, hepatocytes, epithelial cells, keratinocytes, and fibroblasts, as well as other cells...generally in response to initiating stimuli triggered by infection or inflammation". In addition, "many endogenous products result in the release of endogenous pyrogens, thereby causing fever. Such endogenous substances include antigen-antibody complexes, complexes with complement, complement cleavage products, steroid hormone metabolites, bile acids and some cytokines".185 Since "the virus ["HIV"] is replicating 24 hours a day and from day one", 155 and "2x10 9 CD4 + cells [are] produced and destroyed each day", and fever and "many of the associated features of fever can be reproduced by infusions of purified cytokines, including back pain, generalized myalgias, arthalgias, anorexia and somnolence", 185 it is indeed surprising that such "massive" infection and cellular destruction may remain largely, if not totally, asymptomatic for prolonged periods of time in HIV seropositive individuals; (ii) If there is such a "massive" HIV infection, why is it not detected by standard hybridization procedures and why, in order to detect such "massive" infection, did not the authors use PCR which can "amplify a DNA-needle into a DNA-haystack" or even nested PCR but were obliged to determine "Viral RNA" with novel assays, "modified branched DNA (bDNA) or RT-PCR assay and confirmed by QC-PCR" for which no details are given? One of the many problems186,187 associated with the Ho and Wei studies and the methods they employ is illustrated in a presentation at the XIth International Conference on AIDS. Researchers from the Medical School, Camden, New Jersey took a single plasma sample from a patient "with a CD4 cell count of 123 cells/cmm" and divided it into ten aliquots. The RNA from each sample was reverse transcribed and the cDNA "was then amplified with an internal control DNA (mimic) using gag primers...cDNA was also pooled from the initial 10 individual RT reactions and QC-PCR was performed 10 times on pooled cDNA". They reported that "The mean HIV-1 copy number for the 10 individual plasma aliquots was 136,000 RNA copies/ml with a standard deviation of 76,9000 copies/ml (range 74,2000 copies/ml to 334,600 copies/ml). The mean HIV-1 copy number for the pooled cDNA assayed 10 times was 145,900 copies/ml with a standard deviation of 61,900 copies/ml (range 84,500 copies/ml to 259,300 copies/ml)...the RT is not the source of variability in HIV-1 QC-PCR. Rather, variability is likely due to differences in amplification of the target template and internal control used in the QC-PCR assay". 188 According to Maddox and Wain-Hobson both Ho and Wei and their colleagues were able to reach their startling conclusions only after a decade of HIV research because they teamed up with mathematicians and because they were able to use "New techniques for assaying the low levels of virus involved"! (italics ours). It is ironic then that the strongest criticisms of these studies have emanated from mathematicians such as Frank Buianouckas from the Department of Mathematics and Computer Science, City University, Bronx, New York USA and Mark Craddock, School of Mathematics and Statistics, The University of Sydney, Australia. "What is this viraemia of billions of RNA particles that can only be seen with an undocumented branch-PCR or PCR but not with a functional infectivity test?".189 "My question is this: Just what exactly will it take to get people doing HIV research to turn away from high tech, unproven methods, arcane speculations about molecular interactions etcetera etcetera and ask themselves ‘do any of us have the faintest idea what we are doing?’." 190 One can argue that criticism of the Ho and Wei papers by individuals from the HIV/AIDS dissident movement is not to be unexpected but it is unheard of for one group of HIV experts to criticize another as it happened with the Ho and Wei studies.191 In July 1995, as a result of "misgivings" about the claims of Ho and Wei and their colleagues, "two dozen AIDS researchers congregated in Berkeley, California...to challenge the establishment, swap copies of their own manifestos, and enjoy the bonhomie of hanging out for two days with fellow ‘alternative’ thinkers", who concluded that Ho et al and Wei et al "were short on compelling evidence that their ideas were correct".192 (b) According to researchers from the Walter Reed Army Institute of Research, "the extensive use of the polymerase chain reaction (PCR) to recover HIV-1 proviral DNA has favored analysis of the short amplicons that are most efficiently recovered by this technique".193 In fact, in the vast majority of cases the presence of the "HIV genome" is proven by amplifying short "invariant regions" of a "viral gene", usually of the gag gene. However, since it is accepted that a significant proportion of the "HIV genomes" are defective, finding a fragment of a gene is not proof of the existence of the whole gene and even less so for the existence of the whole genome "HIV DNA" or "HIV RNA", a point accepted by many HIV/AIDS researchers. (c) If a unique molecular entity "HIV DNA" exists, then the same primers would be able to amplify it, irrespective of where such unique DNA is found. According to the same researchers, "Due to the extensive genetic diversity of HIV-1, opportunities to identify a single primer pair capable of amplification of diverse subtypes are limited". 193,194 In fact, amplification results obtained with primers for different genes from one subtype are not in complete agreement. For example, in the first "HIV" PCR, two primer pairs to amplify the gag gene were used and it was found that "some samples scored positive with only one of the two primer pairs".195 It is said that in the USA and Europe individuals are almost exclusively infected with subtype B. Yet researchers from the University of Edinburgh found that "The results obtained with the gag and env primers were not in complete agreement. In 5 of the 28 replicates, either the gag or an env sequence was amplified but not both". 182 A PCR study of 40 individuals using primers from the LTR, gag and env regions was performed by French researchers including researchers from the Pasteur Institute. Out of 38 positive samples, "34 were gag positive (90%) whereas env and LTR were detected in fewer cases 24 samples (63%) and 18 samples (47%) respectively...11 of 40 samples were positive with three primer pairs, 16 with two primer pairs and 11 with only one primer pair".196 Such discrepancies may be due to:
(d) No meaningful information can be obtained from a test unless the test is standardized and it is shown to be reproducible. No such data is currently available for the PCR. In fact, since there are so many "HIV" subtypes and one has to use different primers for different sub-types or even for the same subtype, it makes it extremely unlikely that such data can ever be obtained. (e) By far the most important parameter of a test is its specificity, that is, how often a test is negative when the condition sought is absent. For PCR one must have proof that the primers:
No such evidence exists for the "HIV" primers. In fact, since it is not possible to say what the "HIV DNA" sequences are, it follows that it is also not possible to be specific about what the primers represent. Even if one assumes that the "HIV DNA" and thus the primers are specific to a retrovirus since:
it is impossible to say that the "HIV DNA" probes are HIV, or DNA probes of an endogenous retrovirus or even an exogenous retrovirus HTLV-I;
Furthermore, given the facts that: (a) "about one percent of the human genome" consists of endogenous retroviral sequences; (b) homologies exist between the genes of endogenous and exogenous retroviruses, especially in the gag and pol genes, and between these genes and cellular retroelements; specific binding of the "HIV" primers is most unlikely. Even if (i)-(iii) are proven one must still determine the specificity of the PCR reaction, that is, show that no positive results are obtained in individuals who are not infected with HIV. This can only be determined by using HIV isolation as an independent gold standard, that is, by comparing PCR with the procedures listed above (see 6.1). This has not been done, a fact accepted by one of the best known HIV/AIDS researchers, William Blattner "One difficulty in assaying the specificity and sensitivity of human retroviruses [including HIV] is the absence of a final ‘gold standard’". 59 (f) At present some evidence obtained without the use of a gold standard illustrates that the PCR procedure is non-specific:
In a study of 327 health care workers exposed by needlestick injuries to the "human immunodeficiency virus", four had "one or more positive" PCR tests. An additional seven had "an indeterminate PCR test result on the initial specimen". Later samples for all 11 were negative "none seroconverted or developed p24 antigenemia" and "all of the subjects remained healthy".204,205 While the evidence for such occurrence in adults is sporadic, it is much more often reported in children. However, PCR is not used for routine diagnosis of HIV infection in adults and rarely, if ever, is repeated. Unlike in adults, PCR is very often used in children, this being the case because "HIV diagnosis" is "complicated by persistence of passively acquired maternal anti-body". By 1995 numerous studies in children206-209 revealed the conversion of a positive PCR to negative. One of the most recent reports was published in 1995 by French researchers. In a six year cohort of188 "infected" children which was analyzed retrospectively 12 (6.7%) "cleared HIV infection". Each child had at least two positive PCR results at two separate time points in the first year, followed by numerous (up to seven) negative PCR results. For PCR the investigators used primer pairs for the gag, pol, and env gene regions; and the test was considered positive "if at least two genes were amplified". Commenting on their results the authors wrote, "Three different rooms with separate air-conditioned circuits were used for DNA extraction, PCR-buffer preparation, amplification and blotting. Amplicons were never transferred in the area reserved for unamplified sequences. Thus, positive PCR results are unlikely to be due to contamination... Nevertheless, as our PCR assays are performed on unmanipulated cells, culture contamination leading to false positive PCR results is impossible...We therefore consider that the probability of repeated contamination on successive samples from the same child is scarce". The authors "could not find any correlation between either neutralizing or antibody-dependent cellular cytotoxicity-mediating antibodies and HIV clearance". Of 139 children born to HIV positive mothers but who were "clearly negative", "eight were PCR-positive once for a single viral gene (pol), three were positive twice for the pol gene, and once of the three was also positive for the gag gene in a single assay". 210 In 1989, discussing their studies on human retroviruses, researchers from the University of New York wrote, "Irrespective of the origin of human retroviruses, their presence leads to both practical and theoretical concerns. Presently, the major practical concern is that effective use of PCR as a screening procedure for HTLV-I, HTLV-II and HIV infections must always include appropriate controls to ensure that no endogenous sequences contribute to positive signals. As previously noted, HIV unique primers corresponding to the highly conserved reverse transcriptase region shown in Fig. 1 function well in the PCR amplification of HeLa DNA even at annealing temperatures around 60°...Another practical concern is that the use of PCR for determining the possible retroviral etiology of a variety of human diseases may be complicated by endogenous retroviruses. Even if cDNAs are used for PCR templates, the transcriptional activities of endogenous sequences must be considered". 119 In an article published this year, where he discusses the laboratory diagnosis of "HIV infection", Philip Mortimer wrote, "Other diagnostic methods, e.g. p24 antigen testing, and proviral DNA and RNA amplification exist, but these innovations in HIV diagnosis need to be matched against the anti-HIV test and should be rejected unless they fulfil a need that antibody testing fails to meet". 211 According to researchers from the University of London, "The use of polymerase chain reaction (PCR) for the diagnosis of HIV infection is becoming more widespread and although not yet entirely reliable compared with serology, has been of special value in HIV-seronegative intravenous drug users". 200 If PCR needs to be matched against the "HIV" antibody test because it is less reliable than serology then given the fact that at present there is no evidence which shows that a positive "HIV" antibody test is proof of HIV infection, 89 one has no choice but to agree with Shoebridge et al that "until further molecular and biological studies are carried out, it will be unsure as to what detection of HIV-1 DNA, even when shown to be HIV-1 really means". 212 In analyzing the "HIV" molecular biology one cannot help reflecting on the words of Sir John Maddox, "Is there a danger, in molecular biology, that the accumulation of data will get so far ahead of its assimilation into a conceptual framework that the data will eventually prove an encumbrance? Part of the trouble is that excitement of the chase leaves little time for reflection. And there are grants for producing data, but hardly any for standing back in contemplation". 213 CONCLUSION—The present data do not prove the existence of a unique molecular entity "HIV DNA" which constitutes the genome of a unique, externally acquired retrovirus, HIV. Neither is there any proof for the existence of an "HIV quasispecies". Nor is it possible to say what exactly the different "HIV DNAs", the probes and primers derived from these DNAs and the sequences in the cellular DNA with which they hybridise represent. Continued: The Isolation Of Hiv: Has It Really Been Achieved? Part 4 |