The Perth Group answer to Christopher Noble 21 August 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: The Perth Group answer to Christopher Noble

The Perth Group answer to Christopher Noble

In his rapid response “Re: Politics vs. science” (7 August 2003), Christopher Noble wrote: “Eleni Papadopulos-Eleopulos writes (Note that the papilloma virus is a DNA virus yet according to Christopher Noble the human papilloma virus' genome is even more variable than the "HIV" genome, an RNA genome)”. He responds “Please do not twist my words. I stated quite clearly that different isolates of HPV show similar levels of divergence to HIV. At no time did I claim that the mutation rates or evolution rates were comparable. In fact, I specifically stated the opposite. Indeed you previously stated that you were not interested in evolution rates. Why have you changed your mind? Just to refresh your memory. "Neither are we interested in the genomic variation with time, that is, evolution of the genome, but the genomic variation at a given time".

When we wrote “the human papilloma virus’ genome is even more variable then the “HIV” genome”, by variability we mean the “variation at a given time” or “levels of divergence” which Christopher Noble uses and not mutation rates or evolution rates which are changes in time.

Christopher Noble wrote: “You also claimed that the reference I provided regarding HPV (1) was not available in any Australian library. In fact it is. (2)”

Unfortunately our librarian looked only for the availability of hard copies. So we would like to thank Christopher Noble for drawing our attention to its availability in electronic form.

Christopher Noble wrote: “You also completely ignore the further reference I gave for the diversity of HPV. (3) "The overall nucleotide homology to other sequenced HPV types is below 50%. The closest other HPV type is represented by HPV-18, sharing 49% identical nucleotides." “

In his rapid response “A plea to Eleni Papadopulos-Eleopulos” (8 August 2003), Christopher Noble wrote: “Please read the references I have provided regarding HPV.”

Here is our analysis of the references that Christopher Noble listed. In the 1990 paper by Hirt et al (1), the authors claimed to have identified a new “unusual HPV type”. The authors did say that: "The overall nucleotide homology to other sequenced HPV types is below 50%. The closest other HPV type is represented by HPV-18, sharing 49% identical nucleotides." (1) But they also added: “The typical E2 binding sequence ACCN6GGT, found in all papillomaviruses analyzed to date, does not occur in the URR of the HPV-41 genome…A potentially interesting observation relates to the presence of modified presumed E2 binding sites, conserved in all other human pathogenic PVs sequenced thus far. It seems to underline the early separation of HPV-41 type viruses from other HPV types in the course of viral evolution.”(1)

In a paper “Intratype Variation in 12 Human Papillomavirus Types: a Worldwide Perspective” by researchers from the USA, France, Spain and UK published in 1996 (2), the authors wrote: “Human papillomaviruses (HPVs) constitute a group of viruses associated with benign and malignant neoplasia of cutaneous and mucosal epithelia. To date, more than 70 different HPV types have been identified...An HPV genome is defined as a new type if it is separated by a Hamming distance or dissimilarity of more than 10% in its nucleotide sequence compared with other known HPV types in the E6, E7, and L1 open reading frames (ORFs) combined. Isolates within the same type differing by 0 to 2% in their nucleotide sequences compared with the reference sequence are referred to as variants, and those differing by 2 to 10% are referred to as subtypes…PVs are largely dependent upon the host cellular machinery and use the host DNA polymerase for the replication of their genomes. Therefore, it is reasonable to consider that resultant genomic diversity may be constrained similarly for both virus and host. As has been suggested in previous studies, PVs and other persistently infecting small DNA viruses may represent useful tools for studying the coevolution of virus and host. An extension of this suggestion is that the limited polymorphism observed in our data from 12 different HPV types and novel sequences reflects a previously proposed population bottleneck occurring in the host population…Limited information is available concerning HPV intratype variation except, for HPV-6, HPV-11, HPV-16 and HPV-18. Sequence analyses of ORFs, such as E5, E6, E7, L1, and L2, and of the upstream regulatory region (URR) in more prevalent HPVs (e.g. HPV-16 and HPV-18) have indicated that intratype polymorphism is very limited. Intratype variation observed in these HPV types is commonly less than 2.5% intragenically (within ORFs) and 5% extragenically (within the URR). While most sampling and sequencing efforts have been biased toward a search for more divergent HPV genomes, recent investigations have failed to identify intermediate levels of divergence, HPV subtypes have been encountered very rarely…In this study, we have examined intratype human papillomavirus (HPV) sequence variation in a worldwide collection of cervical specimens. Twelve different HPV types including HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM9/PAP238A (recently designated HPV-73), and a novel partial genomic HPV sequence designated MM4/W13B were analysed in this study…Within a single HPV type, nucleotide diversity varied between 0.2 and 2.9% (i.e. between any pair of variants) and the majority of nucleotide changes were synonymous (amino acid conserving)…Presuming that HPVs have evolved under the same constraints as their corresponding hosts, the limited genetic diversity observed for all HPVs studied to date may reflect an evolutionary bottleneck occurring in both virus and host populations…Although data described in our present investigation are limited for many of the HPV types analysed because of small sample sizes, this and other investigations have presented similar values for the intratype pairwise distances for many types of HPVs. Notable is the relative lack of comparisons in the 3 to 10% range and the sharp drop-off around 2.5%. A bottleneck in the human population would have limited the number of HPV lineages that would survive, possibly causing massive extinction of lineages. It is possible that the relative lack of HPV polymorphism in the range between 2.5 and 10% reflects a severe pruning of the HPV evolutionary tree as a result of such a reduction event in the human population approximately 100,000 years ago. Moreover, the relative abundance of variants within the 0 to 2.5% range may reflect subsequent expansion events occurring in the host population about 30,000 years later.”(2)

In the paper by Halpern from the Los Alamos National Laboratory published in 2000 (3), the author found “certain similarities” between SIV/HIV and the papillomaviruses. Comparing HIV with HPVs he wrote: “There are also salient differences. The rates of mutation and/or substitution are remarkably different. This is reflected by differences in temporal and geographic heterogeneity of sequences. There is considerable variation among geographic regions in the prevalence of HIV sub-types (differing from one another by up to 35% on the nucleotide level In contrast, HPV types (differing from one another by more than 10% in L1, and commonly by 30% or more) are more uniformly distributed. Only at the level of the variant (differences between isolates in coding regions generally <3%) is variation in HPV geographic distribution the norm. Likewise, whereas evolution of HIV sequences can readily be observed within a patient and the overall diversity of HIV-1 sequences has increased during the course of the pandemic, HPV isolates have arguably been stable during the period for which we have sequence data. Isolates that are identical over more than 3500 nucleotides have been obtained in different years and different geographic locations. Variants of HPV16 sequenced by Yamada et al (1995) are identical to the corrected sequence of the HPV16 prototype over such lengths. Intra-patient variation within a given HPV type is thought to be rare...Essentially, any degree of divergence may be observed between HIV isolates, although it is necessary to compare epidemiologically linked isolates to see highly similar viruses. There are arbitrarily-many HIV isolates that would constitute new 'types' by the criteria applied to papillomaviruses (> 10% dissimilarity); indeed, any new HIV isolate that is not known to be epidemiologically linked to previous isolates has a good chance of satisfying these criteria. It is the norm for two clones from a single HIV isolate to show at least some variation. In contrast, within the HPVs, most types show relatively small ranges of divergence around the consensus pattern. Furthermore, it is not generally possible to find large numbers of new types within an extensively studied group...Repeated sampling from an individual reveals stability of the genome...The rate of evolution in HIV is among the fastest ever observed…In contrast, the evolution of papillomaviruses appears to be extremely slow, in keeping with the use of host enzymes to replicate and proofread the viral genome. As already noted, HPV genotypes are quite stable over both time and space…As already noted, there are geographic differences in HPV variant distribution, much as there are differences in the distributions of HIV-1 subtypes. As for HIV, a wide range of divergent HPV variants can be found in Africa. However, the interpretation of these distributions may be quite different. In the case of HIV, they are widely interpreted as the result of founder effects in a recent pandemic that began in Africa. In the case of PV, it may instead reflect a very ancient family of viruses that came out of Africa with the spread of modern humans around the globe...Such a dating of the age of this point mutation would suggest that the more divergent variants, differing by as much of 5% in this region of the genome, are considerably older, perhaps in the order of 100,000 years…Bernard and coworkers have given several grounds for assuming that the major clades (supergroups) of papillomaviruses are ancient…With a virus such as HIV, the level of diversity within an individual at any given time is substantial, the level of diversity within the host population is enormous…With papillomaviruses, the situation is of course quite different. Although the maximum differences between HPV types are as great as between any pair of HIV/SIV sequences, there appears to be a finite number of types, subtypes and even major variant branches. Moreover, a small fraction of this number is associated with the majority of the disease burden. The mutation rate and rate of virus production, while not known exactly, are clearly much lower than for HIV…In contemplating a vaccine for human papillomaviruses (HPVs), it is important to consider the evolutionary context in which such a vaccine would be deployed. The human immunodeficiency virus, having been the subject of even more extensive study than HPV, shares certain salient features with regards to phylogenetic structure, and may serve as a model for contemplation of possible difficulties with HPV vaccination. However, there are also striking differences in the evolutionary potentials and histories of the viruses that permit an optimistic outlook for HPV.”(3)

In other words: While "HIV-1" is said to be a unique virus, the HPVs are "a very ancient family of viruses". ("Virus families represent groupings of genera of viruses that share common characteristics and are distinct from the member viruses of other families…Most of the families of viruses have distinct virion morphology, genome structure, and/or strategies of replication, indicating phylogenetic independence or great phylogenetic separation. At the same time, the virus family is being recognised as a taxon uniting viruses with a common, even if distant phylogeny"). (4)

While “HIV-1” is said to be a new human virus transmitted in the last few decades from animals, the HPVs existed in humans since ancient times. While there is a limited number of HPV types (about 70), that is viruses which have "dissimilarity of more than 10%), "There are arbitrarily-many HIV isolates that would constitute new 'types' by the criteria applied to papillomaviruses (> 10% dissimilarity); indeed, any new HIV isolate that is not known to be epidemiologically linked to previous isolates has a good chance of satisfying these criteria".

While the present dissimilarities in the "HIV-1" genome appeared at the time of its transmission to humans (a few decades ago), the present dissimilarities in the HPV types existed since the ancient times. While the "HPV genotypes are quite stable over both time and space", there is "an extraordinary scale of HIV variation" both in time and space (5).

While "HIV-1" (all groups) is said to always cause a fatal syndrome, AIDS, in all infected individuals, only "a small fraction" of the HPV types are associated with "disease burden". Furthermore, "After the plurality of HPV types was disclosed, distinct types of HPVs were shown to be associated with various warts differing by characteristic morphology and histologic features". (6) "Results of HPV typing by Southern blot of specimens from 2627 women in eight studies were collated and analysed. A descriptive analysis defined four categories: low risk (HPV 6, 11, 42, 43, 44) detected in 20% of all low grade histological cervical lesions but none of 163 cancers, intermediate risk (HPV 31, 33, 35, 51, 52, 58) detected in 2% of high grade squamous intraepithelial lesions (HSIL) but only 10% of cancers, high risk/HPV 16 found in 47% of HSIL and cancers, and high risk/HPV18 associated with 27% of cancers but only 7% of HSIL…Another influential paper at this time that reinforced the concepts of low risk and high risk was published by Van Ranst et al. Phylogenetic trees based on nucleotide alignments of HPV genes were constructed. Quite distinct groups of HPVs emerged, including groups referred to as mucosal low risk (HPV 6, 11, 13, 42, 43, 44) and mucosal high risk (HPV 16, 18, 31, 3, 5, 39, 45, 51, 52, 56, 58, 59, 68 and 83)". (7)

Christopher Noble wrote: “You have repeatedly claimed that "the genomes of the most variable RNA viruses do not differ by more than 1%". I will ask you once more to provide evidence for this claim.”

The reference in which we said it is stated that even a 1% difference is considered to represent “extreme heterogeneity of viral populations” (8) was given before in our rapid responses “”HIV” genome, clones and sequences” (18 July 2003) and “The “HIV” and influenza A virus genomes” (26 July 2003).

Christopher Noble wrote: “You also quote Korber et al (6) "A phylogenetic tree of HA sequences sampled world-wide in 1996 shows much less diversity than a sampling of subtype B HIV-1 envelope sequences from a single city, Amsterdam in 1990-1991." You forgot to also quote " Tree based on 96 HA1 domain sequences of human influenza H3N2 viruses". You have previously compared the most divergent HIV isolate HIV-1 group O. You should therefore compare H3N2 isolates and H1N1 isolates form the same year.”

As we have already stated in our rapid response “Re: Politics vs. Science” (5 August 2003) “It is true that a difference of up to 40% exists between HIV-1 M group subtype B and HIV-1 O group and HIV-1 group N but also within the HIV-1 M group.” This can be seen in references (1, 2) that we gave. In other words, we did not compare only “the most divergent HIV isolate HIV-1 group O”. (5, 9) According to the researchers from the Los Alamos National Laboratory in a given year there is “a relatively homogenous viral population” of influenza”.(5)

Christopher Noble wrote: “Now tell the difference between these isolates with electron microscopy.”

It is true that with electron microscopy you cannot tell the nucleic acid sequence differences “between these isolates”. But, it is equally also true that the nucleic acid sequences by themselves do not tell you that they represent the genome of a retrovirus. The minimum requirement to claim that they represent the genome of a retrovirus is to have electron microscopy proof that their ultimate origin was a retrovirus-like particle.

Christopher Noble wrote: “Let's go back to your original argument for dismissing thousands of papers describing the HIV genome. 'In other words, if a mere difference of less than 2% leads to the appearance of two totally different objects (namely, humans and chimpanzees), how then can differences of up to 40% lead to the appearance of the same object'. According to your argument neither HPV nor influenza exists because divergent isolates show differences of much more than 2%. Will you deny the existence of HPV and influenza or will you admit that your previous arguments were deceptive and misleading?”

In his rapid response “A plea to Eleni Papadopulos-Eleopulos” (8 August 2003), Christopher Noble wrote: “According to your arguments HPV and the influenza virus do not exist. You have used this argument as an excuse to totally ignore thousands of papers on the HIV genome. Why is it applicable to HIV and not to HPV and influenza?” You have been arguing that thousands of scientists are wrong about the HIV genome”.

We are amazed that Christopher Noble has ignored all our main arguments concerning the evidence which is said to prove the existence of “HIV”. Instead, he has solely concentrated on one of our minor arguments, that is, the genomic differences. Let us make it plain: (i) the existence of one, two or an infinite number of viruses cannot be used as proof for the existence of another virus. (ii) if all the cultures containing tissues from AIDS patients or even if all the AIDS patients and nobody else contained a unique fragment of RNA, that is, a fragment of the same length and no sequence variability whatsoever, it does not constitute proof that this RNA (cDNA) is the genome of a unique retrovirus or any other virus.

We have not ignored the “thousands of papers on the HIV genome”. To the contrary, we have analysed as many as we could but we could not find in any of them proof for the existence of the “HIV” genome. It is possible that we may have missed some. Would Christopher Noble please provide a few of the thousands of references that would answer our requests so we may be enlightened about the proof for the existence of the “HIV” genome?

Christopher Noble wrote: “The least you could do is answer some of my questions”.

It is our view that we have been answering Christopher Noble and everyone else who has put questions to us, paragraph by paragraph. Would Christopher Noble please do the same, starting with our requests in our previous rapid response “A plea to Christopher Noble” (8 August 2003)? These requests are as follows:

(a) A few references which prove that the “purified HIV”, that is, the 1.16gm/ml band from which the “HIV” genome and proteins originated, contain particles in which “No apparent differences in physical appearance could be discerned” and the particles have the morphology of retroviruses.

(b) A few references with proof that: (i) the molecules used in cloning of a complete viral (“HIV”) genome originated from “HIV” particles; (ii) the “HIV” genetic sequences” originated from “HIV” particles.

(c) A few references which prove that the “HIV” antibody tests are specific. To claim proof for specificity there MUST BE at least one study and a few confirmatory studies where the antibody antigen reaction (assuming that the antigens are HIV) is compared with the present or absence of “HIV”, that is, with “HIV” isolation/purification. This study must include a statistically significant number of both patients who have AIDS as well as patients who do not have AIDS but are sick. In addition, the test must be done blind.

References 1. Hirt L, Hirsch-Behnam A, de Villiers EM. Nucleotide sequence of human papillomavirus (HPV) type 41: an unusual HPV type without a typical E2 binding site consensus sequence. (1991) Virus. Res. 18: 179-89.

2. Stewart ACM, Eriksson AM, Manos MM, Munoz N, Bosch FX, Peto J, Wheeler CM. Intratype Variation in 12 Human Papillomavirus Types: a Worldwide Perspective. (1996) J. Virology 70(5): 3127-3136.

3. Halpern AL. Comparison of papillomavirus and immunodeficiency virus evolutionary patterns in the context of a papillomavirus vaccine. (2000) J. Clinical Virology 19:43-56.

4. The Universal System of Virus Taxonomy. http://www.ncbi.nlm.nih.gov/ICTV/int ro_to_universal/universal_system.html

5. Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, Detours V. (2001). Evolutionary and immunological implications of contemporary HIV-1 variation. British Medical Bulletin 58: 19-42.

6. Majewski S, Jablonska S. The role of HPVs in benign and maligant cutaneous proliferations. (2003) Papillomavirus Report 14(1): 1-10.

7. Lacey CJN, Roman A, Brown DR. Low oncogenic risk anogenital HPV infection. (2002) Papillomavirus Report 13(4): 103-109.

8. Steinhauer DA, Holland JJ. Rapid evolution of RNA viruses. (1987) Annual Review of Microbiology 41:409-433.

9. Kozal MJ, Shah N, Shen N, Yang R, Fucini R, Merigan TC, Richman DD, Morris D, Hubbell E, Chee M, Gingeras TR. (1996). Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays. Nat Med 2:753-759.

Competing interests: None declared