More On Genomic Variability 23 October 2003
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Eleni Papadopulos-Eleopulos,
Biophysicist
Department of Medical Physics, Royal Perth Hospital, Western Australia,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso

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Re: More On Genomic Variability

More On Genomic Variability

In his rapid response “Re: Genomic Variability” (14th October 2003), Christopher Noble wrote: “Eleni Papadopulos-Eleopulos writes: "Again, suppose for the sake of argument that there are published papers with evidence (and not merely evidence in databases) which prove that in RNA viruses the nucleic acid "sequences differ by approximately 50%" ...... " There is no need for supposition. Within influenza A subtypes, hemagglutinin sequences differ by up to 20%; between subtypes up to 60%. (1) Why do you continue to deny this?“

In his reference 1: (i) the authors compared not only human influenza A viruses but also influenza A viruses of birds obtained at different times from around the world (A/Turkey/Ontario/6118/68, A/Duck/Alberta/60/76, A/Gull/Maryland/704/77, A/Duck/Czechoslovakia/58, A/Shearwater/Australia/1/72, A/Turkey/Wisconsin/66, A/Duck/England/56, A/Duck/Ukraine/1/63, A/Tern/South Africa/61) (This is the same as comparing “HIV-1”, “HIV-2”, “SIV” and other animal immunodeficiency viruses such feline); (ii) the sequence differences were based upon several assumptions.

Christopher Noble wrote: “I asked you a specific question regarding the genomes of the Sabin polio strains. You have not answered. Why?”

In his rapid response “The Perth Group “answer” to Christopher Noble” (5th September 2003) Christopher Noble asked: “Do the three Sabin polioviruses differ from each other by more than 1%? Yes or No?” and gave us several databases to look up. Our answer was given in our rapid response “Finding wood among the trees” (22nd September 2003): “(a) it is not sufficient to just look at databases and phylogenetic trees of influenza A, papilloma, polio viruses, but one has to determine the origin of the sequences and the method used in phylogenetic analysis; (b) we are not interested in these viruses and the topic of this debate is “HIV”.”

Since the topic of this debate is still “HIV”, it is not sufficient to just look at databases and phylogenetic trees and the existence/non- existence of a given virus cannot be either proven or disproven by another virus, our answer remains the same.

Christopher Noble wrote: “Eleni Papadopulos-Eleopulos continues: "The problem then is how loose the "leash" can be before you start asking questions in regard to: (i) protein function.." It is your argument. Why don't you tell us? You are the one that has been saying that "the genomes of RNA viruses do not differ by more than 1%" and attempting to use this "fact" to argue against the existence of HIV.”

Let us again reiterate that the 1% variability is not our claim but that of a number of virologists.

Regarding how loose the leash can be, we felt that Christopher Noble would consider more readily the views of eminent virologists such as Robert Gallo, Peter Duesberg, Esteban Domingo and researchers from the Pasteur Institute rather than ours. That is why we gave their views in our rapid response “Genomic Variability” (14th October 2003). If Christopher Noble disagrees with their views, perhaps he may try to convince them to change their views and rewrite virology.

Christopher Noble wrote: “The hemagglutinin gene in different influenza A subtypes differ by approximately 50% and yet the protein performs the same function in the virus. In fact, reassortment is a basic feature of influenza A evolution. When a host is infected with two different types of influenza A the hemagglutinin gene of one virus can be swapped with the hemagglutinin gene of another. The hemagglutinin genes are interchangeable. They perform the same function.”

The title of his reference 1 is “Comparison of Complex Amino Acid Sequences and Receptor-Binding Properties among 13 Serotypes of Hemagglutinins of Influenza A Viruses”. In this paper the authors wrote: “The hemagglutinin (HA) glycoprotein of influenza A virus performs two crucial functions in the early stage of virus infection. First, HA is responsible for binding of the virus to cell surface receptors and second, it mediates liberation of the viral genome into the cytoplasm through membrane fusion…Receptor-binding specificity was assayed with gangliosides containing sugar chains with either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc) and with either NeuAcá2,3Gal or NeuAcá2,6Gal linkage for the HA of human and avian origin, and of various serotypes (H1 through H10 with the exception of H7)…It is worth noting that A/PR/8/34 (H1N1), A/Duck/Ukraine/1/63 (H3N8), A/Duck/Czechoslovakia/56 (H4N6), A/Tern/South Africa/61 (H5N3), A/Turkey/Ontario/6118/68(H8N4), and A/Chicken/Germany “N”/49 (H10N7) preferentially bound to NeuAcá2,3Gal-containing oligosaccharides, whereas, A/Japan/305/57 (H2N2), A/Aichi/2/68 (H3N2), A/Shearwater/Australia/1/72(H6N5), and A/Turkey/Wisconsin/66(H9N2) bound to NeuAcá2,6Gal-containing oligosaccharides.” (1)

In the same paper, one reads: “amino acid sequence homology within a serotype is 80% or more even between virus strains readily distinguishable by polyclonal antibodies…substitution of a single amino acid out of five to six residues which usually make up an antigenic determinant may profoundly alter the antigenicity…”. (1)

So protein variability does lead to functional and antigenic variations. That this is the case, one need look no further than the sickle cell syndromes.

Christopher Noble wrote: “Your argument concerning the genetic variation of RNA viruses is demonstrably false. For some reason you refuse to admit this.”

Our argument is based on the views of eminent virologists. Christopher Noble also wrote: “ Within influenza A subtypes, hemagglutinin sequences differ by up to 20%; between subtypes up to 60%.” In his rapid response “HIV genomic variations” (5th September 2003) Christopher Noble wrote: “two subtypes of hemagglutinin from influenza A, isolated from humans at approximately the same time, differ from each other by 81% at the amino acid level.”

Christopher Noble may be able to convince these virologists that amino acid polymers having variations of 60%, 81% or even 100% represent the same protein which perform the same function, can be detected by the same antibody test, can be considered to be the same constituent of a viral particle which can be neutralized by vaccines to this protein. He may also be able to convince them that if there is a 60% change in their proteins, they will still be Peter Duesberg, Robert Gallo, Esteban Domingo, Wain-Hobson, they will still look the same and perform the same functions. Then he may be able to rewrite not only virology and molecular biology but also biology.

Christopher Noble wrote: “Eleni Papadopulos-Eleopulos continues: "Since the human genome contains endogenous retroviruses which can be expressed especially in cultures under the conditions used by "HIV" experts, Christopher Noble then like us will conclude that such viruses may not be exogenous and in fact may not be present in the AIDS patients." There is very little sequence similarity between human endogenous sequences and HIV sequences. Only short matches of around 18 base pairs can be found - a tiny fraction of the HIV genome. The same sort of matches can be found if we compare an influenza A genome and the human genome. For some reason you do not seem to be claiming that "influenza" virus sequences are endogenous. I presume this is because you do not have a competing theory for the causation of influenza.”

We don’t know how Christopher Noble found only around 18 base pairs similarities between human endogenous sequences and “HIV” sequences. A few examples will illustrate that this is not the case: (i) 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 hybridisation with HIV probes.(2) Note that Weiss never claimed that this isolate was either “HIV” or another exogenous retrovirus. (ii) DNA extracted from thyroid glands from patients with Grave's disease hybridises with "the entire gag p24 coding region" of “HIV”.(3) (iii) 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 5 patients without HIV-1 infection" were examined using an HIV gag probe. "The antisense riboprobe hybridized to cells known to be infected with HIV-1. It hybridised 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".(4) The authors concluded that there is a neurone-specific 9.0- kb transcript that shows extensive homology with antisense gag “HIV-1” sequences and that this transcript is expressed in neuronal cells of both “HIV-1”- infected and noninfected individuals. (iv) 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".(5) (v) In a 1993 study, "In one kidney recipient (the donor was negative for p24 antigen) who, 3 days following transplantation developed fever, weakness, myalgias, cough and diarrhoea, all "Bacteriological, parasitological and virological samples remained negative. The only positive result was antigenaemia p24, positive with Abbot antigen kits in very high titers of 1000pg/ml for polyclonal and 41pg/ml for monoclonal assays. This antigenaemia was totally neutalizable with Abbot antiserum anti-p24...2 months after transplantation, all assays for p24-antigen became negative, without appearance of antibodies against HIV. Five months after transplantation our patient remains asymptomatic, renal function is excellent, p24 antigenaemia still negative and HIV antibodies still negative".(6) Using two kits, the Abbot and Diagnostic Pasteur, in one study, p24 was detected transiently in 12/14 kidney recipients. Peak titres ranged from 850 to 200 000 pg/ml 7-27 days post- transplantation. Two heart and 5/7 bone marrow recipients were also positive, although the titres were lower and ranged from 140-750 pg/ml. Disappearance of p24 took longer in kidney (approximately 6 months) than in bone-marrow (approximately 4-6 weeks) recipients. Discussing their findings the authors wrote: "The observation of a 25-30kD protein binding to polyclonal anti- HIV human sera after immunoblots with reactive sera raises several questions...The 25-30kD protein could therefore be compared with the p28 antigen recently described with human T-cell- related virus lymphotropic- endogenous sequence...The characterization of this 25-30kD protein may represent an important contribution to the detection of HIV-1-related endogenous retroviruses".(7)

Christopher Noble wrote: “If you want to you can purchase the cell lines, such as H9 or HUT78 etc., used for growing HIV. (2) Now, "oxidise" these cells with PHA or whatever you want and get them to produce RNA that has even vaguely significant sequence similarity to HIV RNA. If you could get a sequence with open reading frames for gag,pol,vif,vpr,vpu, tat,rev,env,nef there might be a Nobel prize for you. The world is eagerly awaiting your contribution to science.”

It is common knowledge that although initially Gallo implied that H9 was an original cell line developed in his laboratory, it is nothing more than a clone of HUT-78. Since Gallo and his associates were the first to introduce the “HIV” genes, we would be grateful if Christopher Noble reads their papers (8,9,10) and tell us what evidence presented in these papers proves beyond reasonable doubt the existence of the genome of a unique retrovirus and of its genes “gag,pol,vif,vpr,vpu, tat,rev,env,nef”.

As far as experiments are concerned the Perth Group has repeatedly proposed the need for experiments as well as actual experiments. Regrettably both lack of money and obstruction by our protagonist colleagues and others have so far prevented these from taking place.

References

(1) Nobusawa,E., Aoyama,T., Kato,H., Suzuki,Y., Tateno,Y. and Nakajima,K. (1991) Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology, 182: 475-485.

(2) Webster ADB, Dalgleish AG, Beattie R, et al. Isolation of retroviruses from two patients with "common variable" hypogammaglobulinemia. Lancet 1986;i:581-582.

(3) Ciampollio A, Marini V, Buscema M. (1989) Retrovirus-like sequences in Grave's disease: Implications for human autoimmunity. Lancet i:1096-1100.

(4) Wu TC, Kanayama MD, Hruban RH, Whitehead W, Raj BK. (1993) Detection of a neuron-specific 9.0-kb transcript which shares homology with antisense transcripts of HIV-1 gag gene in patients with and without HIV-1 infection. Am J Pathol 142:25-31.

(5) Horwitz MS, Boyce-Jacino MT, Faras A. (1992) Novel human endogenous sequences related to human immunodeficiency virus type 1. J Virol 66:2170- 2179.

(6) Vincent F, Belec L, Glotz D, Menoyo-Calonge V, Duboust A, Bariety J. (1993) False-positive neutralizable HIV antigens detected in organ transplant recipients. AIDS 7:741-742.

(7) Agbalika F, Ferchal F, Garnier JP, Eugene M, Bedrossian J, Lagrange PH. (1992) False-positive HIV antigens related to emergence of a 25-30kD proteins detected in organ recipients. AIDS 6:959-962.

(8) Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong-Staal F. Molecular cloning and characterization of the HTLV-III virus associated with AIDS (1984) Nature 312: 166-169.

(9) Shaw GM, Hahn BH, Arya S, Groopman JE, Gallo RC, Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. (1984) Science 226: 1165-1171.

(10) Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, Gallo RC, Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277-284

Competing interests: None declared