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HIV TRANSMISSION FROM CHIMPANZEES

In their letter to Nature (February 4th), Gao et al claimed to have proven;

 (i)         the identification in a chimpanzee of the genome of a new simian immunodeficiency virus (SIVcpz), SIVcpz US

(ii)        that their chimpanzee, Marilyn, as well as two of the other 3 chimpanzees in which to date a SIVcpz was reported, GAB1 and GAB2, belonged to a chimpanzee subspecies, P.t. troglodytes

(iii)       "the natural range of P.t. troglodytes coincides uniquely with areas of HIV-1 group M, N and O endemicity"; 

(iv)       "HIV-1 infection of humans occurred as a result of cross-species transmission of SIVcpz from P.t. troglodytes". 

(v)        "P.t. troglodytes is the primary reservoir for HIV-1"; 

A close analysis of the evidence on which they are basing their claims raises several questions: 

(a)        Marilyn was "wild-caught in Africa (country of origin unknown), exported to the United States as an infant".1 

            Two of the chimpanzees, GAB1 and GAB2, originated in Gabon.  GAB1, who was 4 years old when reported HIV-1 positive, was caught when she was "about 6 months" and was kept with another 49 wild-caught animals at the International Centre of Medical Research (CIRM) in Gabon.  GAB2, who was also reported HIV-1 positive, was about 2 years old when she was shot in the wild, kept in a village for 2 days and then was brought to CIRM, "where it died of its wounds one week later".2 

            In the 1989 study where the "HIV-1 seropositivity" of GAB1 and GAB2 was reported, the authors (which included one of Gao's co-authors, Peeters) concluded:  "...on examination, none of the people caring for the animals and none of those living in the village showed antibodies to HIV/SIV.  Furthermore, the region where the chimpanzee was captured is known to have a low seroprevalence rate...It has been suggested that human AIDS retroviruses originated from monkeys in Africa.  However, this study and other previous studies on SIV do not support this suggestion".  In other words, by the time when "HIV infection" and AIDS already reached their peak in the US, Europe and Australia, the number of individuals proven HIV seropositive in Gabon were few if any. 

            In a 1990 study published in Nature by researchers from CIRM and the Pasteur Institute, including Wain-Hobson, where the authors described "the molecular cloning and sequencing" of SIVcpzGAB1, one reads:  "In Gabon, only 2 out of 83 chimpanzees tested were seropositive, indicating that SIVCPZ is not widely dispersed in this region...Of more than 250 chimpanzees caught over the last 15-20 years in West Africa, none were seropositive.  This might explain the absence of naturally infected chimpanzees in captivity in the United States as virtually all are of West African origin".3 

How is it possible to espouse "prevalence in the natural host "geographic coincidence", and "plausible routes of transmission" as evidence to substantiate the claims that HIV-1 originated in P.t. troglodytes and that this sub-species is the natural reservoir for HIV-1? 

(b)       The three P.t. troglodytes, GAB1, GAB2 and Marilyn were said to be infected with HIV-1/SIVcpz on the basis of an antibody test. 

However given that: 

(i)         as Philip Mortimer points out, “…it may be impossible to relate an antibody response specifically to HIV-1 infection”.4

(ii)        when the blood was collected none of the animals was perfectly healthy although none had AIDS.

(iii)       the only way to prove the specificity of an antibody test is to use the virus isolation as a gold standard.  Although no effort has been spared, no SIVcpz could be isolated either from GAB2 or Marilyn (see comments below for GAB1).  

How is it possible to claim proof for infection on the basis of an antibody test?

(c)       

(i)         If GAB1 and Marilyn were infected then, given that the animals were brought to the colony as infants where no other animals or humans working there were infected and, according to Weiss, "Chimpanzees in captivity are mostly taken from the wild before they become sexually active and so rarely harbour SIV", how did these two chimpanzees become infected?5 

(ii)        Since the three chimpanzees found positive were all female, and since HIV/SIV is acquired following sexual maturity, how did they become infected? 

(iii)       If the animals were infected with a virus SIVcpz and this was transmitted to humans, why was this not transmitted to any other of the 49 animals at CIRM where GAB1 was kept or to the 93 animals in the colony where Marilyn was kept, not even to her 6 living offspring or her mates?  (By the age of 26 she had a total of 14 pregnancies).6 

(d)       The additional "lines of evidence" that Gao et al use to substantiate transmission are based on genomic studies.  Gao et al claimed to have shown that "All HIV-1 strains known to infect man, including HIV-1 groups M, N and O, are closely related to just one of these SIVcpz lineages, that found in P.t. troglodytes".  Indeed, if all these HIV-1 and SIVcpz strains represented one and the same virus, then their genomes will have to be "closely related".  In fact they should represent a unique molecular entity.  Even in the genomes of RNA viruses, including influenza, which are considered to be most variable, a 1% sequence difference is considered to represent "extreme variability".7  This is because small genetic differences lead to significant phenotypic differences.  For example the difference between the human and the chimpanzee genome is less than 2%. 

            In the 1989 study of SIVcpzGAB1 Peeters et al wrote:  "Nucleic acid hybridisation experiments appear to indicate that the virus is different from HIV-1 and HIV-2".  In the 1990 Nature paper by researchers from CIRM and the Pasteur Institute, including Wain-Hobson, one reads:  "Several regions of the chimpanzee sequences were more than 50% divergent with respect to HIV-1BRU.  Some parts of the gag gene were almost as varied as the hypervariable regions in env...The vpu gene found only in the type 1 viruses was particularly different (64% divergent to HIV-1BRU)…It is also apparent that the SIVCPZ genome was not simply a more diverged HIV-1 isolate…It is not possible to conclude that SIVCPZ was the precursor to HIV-1, if indeed infection ever passed in that direction.  Even given this premise the vpu data indicates that SIVCPZ  was not the immediate precursor".3           

In a 1994 study of the SIVcpzGAB2, Peeters and his colleagues wrote:  "The genetic distance between SIVcpz-gab [SIVcpz GAB1] and SIVcpz-gab2 is 14.1%.  Genetic distances to the HIV-1 genotypes A, B and D strains are 13.7 to 16.3%, whereas distances to group O HIV-1 strains are 15.4 to 18.5%".  Contrary to Gao, in 1994 Peeters and his associates concluded:  "On the basis of their respective distances to each other and to the HIV-1 strains SIVcpz-gab and SIVcpz-gab2 can be assigned as representative for two distinct genetic lineages of HIV-1-related chimpanzee lentiviruses".8  By 1993 it was 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".9 

The HIV-1 group O has "65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences.  The env gene of MVP-5180 [HIV-1 group O] had similarities to HIV-1 and HIV-2 of 53 and 49% respectively...Comparison of the MVP-5180 amino acid sequences with that of the Gabon chimpanzee virus showed similarities of 70, 78 and 53% in the gag, pol and env genes, respectively".10 

As far as the genomic differences between HIV-1 group N, on the one hand, and group M and O on the other is concerned, it suffices to quote from the 1998 study where its existence was first reported.  "Proviral DNA amplification with several sets of HIV-1 group M and O primers was attempted on pelleted end-cultured cells.  Amplification was negative with eight different group M env, gag or pol primers and five group O env or gag primers".11  How is it possible to claim proof for the existence of a unique molecular entity which constitutes the genome of a unique retrovirus HIV-1/SIVcpz? 

(e)       The only way to prove that an RNA (and its cDNA) is the genome of a retrovirus is to demonstrate that it comes from a retrovirus particle and such RNA codes for its proteins.  This can be done only by obtaining the particles separate from everything else, purifying, isolating them.12 

In the 1989 study where Peeters et al reported the isolation of SIVcpzGAB1, stimulated peripheral blood lymphocytes "from healthy human donors" were cocultured with the same type of cells from the chimpanzees.  Supernatant from the coculture was centrifugated for 10 mins at 400.000g.  Detection in the pellet of reverse transcriptase activity, using An(dT)12-18 as template primer was considered proof for SIVcpzGAB1 isolation.  Such a method for viral isolation is no different from claiming that elevations in serum liver enzymes proves the existence of gallstones and moreover, that the gallstones have been isolated from the patient and are in the surgeon’s hands separate from everything else.  The SIVcpzGAB1 "genome" was obtained either by hybridising the RNA present in the pellet (they presented no proof that the pellet contained even retrovirus-like particles), where one would expect to find ample cellular RNA, with probes "from HIV-1oyi, a Gabonese HIV-1 strain", or from "SIVCPZ-infected human lymphocytes" again using HIV-1oyi as a probe.  The "genome" thus obtained was compared with the genome of HIV-1BRU

We could find no details as to how the HIV-1oyi "genome" was obtained.  HIV-1BRU is the "HIV-1" which according to Weiss was "discovered by Barre-Sinoussi and her colleagues in 1983".  The senior author of the 1983 Barre-Sinoussi study was Luc Montagnier.  However, in 1997 Montagnier not only acknowledged that they did not isolate HIV-1BRU, but their "pure" virus from where they chose some RNA and called HIV RNA, did not even contain particles with "morphology typical of retroviruses".13 

The only evidence ever presented as proving the existence of the SIVcpzGAB2 genome was reported by Peeters and his associates and is as follows:  "From this chimpanzee we have been unable this far to isolate a lentivirus, but some of the primary peripheral blood mononuclear cells (PBMCs) have remained available in a frozen state.  To investigate the genetic relationship to the SIVcpz-gab isolate [SIVcpzGAB1], proviral DNA was extracted from these primary PBMCs [no mention is made how it was possible to extract the proviral DNA from the chimpanzee DNA], and a 280-base pair (bp) fragment of the pol gene was amplified by a nested polymerase chain reaction (PCR).  Subsequently PCR fragments were cloned and sequenced".8  No mention is made as to how they obtained the PCR primer.  Gao et al used "consensus sequences" as primers and the following method:  "Here we used the polymerase chain reaction (PCR) to amplify HIV- or SIV-related DNA sequences directly from uncultured (frozen) spleen and lymph-node tissue obtained at autopsy in order to characterise the infection responsible for Marilyn's HIV-1 seropositivity.  Amplification and sequence analysis of subgenomic gag (508 base pairs (bp)) and pol (766 bp) fragments revealed the presence of a virus related to, but distinct from, known SIVcpz and HIV-1 strains.  Because virus isolation from the autopsy tissues was unsuccessful, we used PCR to amplify and sequence four overlapping subgenomic fragments that together comprised a complete proviral genome, which we termed SIVcpzUS". 

However,

(i)        The specificity of the PCR for HIV has never been proven.  The only way to obtain such proof is to use virus isolation as a gold standard.  Even if one accepts the claims for SIVcpzGAB1 isolation, it is agreed that although no effort has been spared SIVcpz could not be isolated from the other two animals.  This means that the PCR results obtained for the genomes of SIVcpzGAB2 and SIVcpzUS are false.

(ii)        Even if they were specific for retroviruses; given that:

(a)       The genome of all human beings and animals contain retroviral proviruses i.e. genomes of the endogenous retroviruses.14

(b)       There are homologies between the genomes of different retroviruses especially in the gag and pol genes.  In fact according to Montagnier and Wain-Hobson the gag and pol genes "are generally conserved among retroviruses".15

(c)       In not one of the studies which claimed proof for the existence of the SIVcpz genomes did the authors use controls. 

(iii)       How is it possible to claim that the sequences detected in the DNA "of SIVcpz-infected human lymphocytes", the PBMCs of GAB2 and in Marilyn's "spleen and lymph-node tissue" were those of an exogenous retrovirus which is transmitted from one chimpanzee to another and from chimpanzee to humans and not those of an endogenous retrovirus? 

(iv)      Since there is no proof that the three chimpanzees ever came in contact with HIV-1 infected humans or animals or that they transmitted such a virus to other humans or animals, is it not more "plausible" to conclude that if these animals did harbour a retrovirus, the retrovirus was endogenous? 

In analysing the "SIVcpz" molecular biology one cannot help reflecting upon 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".16 

Eleni Papadopulos‑Eleopulos1  Valendar F.Turner2  John M. Papadimitriou3  David Causer1  Bruce Hedland-Thomas1  Barry Page1 Charles Geshekter4 Etienne DeHarven5

1Department of Medical Physics, Royal Perth Hospital, Perth, Western Australia. Email vturner@westnet.com.au  2Department of Emergency Medicine, Royal Perth Hospital, Perth, Western Australia; 3Department of Pathology, University of Western Australia.  4Department of History California State University, Chico Chico, California USA  5Professor (Emeritus) of Pathology, University of Toronto, Toronto, Canada.

REFERENCES 

1.         Gao, F., et al. Nature 397, 436-441 (1999).

2.         Peeters, M., et al. Aids 6, 447-51 (1992).

3.         Huet, T., Cheynier, R., Meyerhans, A., Roelants, G. & Wain-Hobson, S. Nature 345, 356-9 (1990).

4.         Mortimer, P.P. Med. Internat. 56, 2334-2339 (1989).

5.         Weiss, R.A. & Wrangham, R.W. Nature 397, 385-386 (1999).

6.         Gilden, R.V. et al. Lancet i, 678-679 (1986).

7.         Steinhauer, D.A. & Holland, J.J. Annual Review of Microbiology 41, 409-433 (1987).

8.         Janssens, W., et al. AIDS Research and Human Retroviruses 10, 1191-2 (1994).

9.         Louwagie, J., et al. AIDS 7, 769-780 (1993).

10.       Gurtler, L.G., et al. Journal of Virology 68, 1581-5 (1994).

11.       Simon, F., et al. Nature Medicine 4, 1032-7 (1998).

12.       Toplin, I. Spectra , 225-235 (1973).

13.       Tahi, D. Continuum 5, 30-34 (1998).

14.       Lower, R., Lower, J. & Kurth, R. Proceedings of the National Academy of Sciences of the United States of America 93, 5177-5184 (1996).

15.       Wain-Hobson, S., Alizon, M. & Montagnier, L. Nature 313, 743 (1985).

16.       Maddox, J. Nature 335, 11 (1988).

 

Reference 13 http://www.virusmyth.com/aids/data/dtinterviewlm.htm