I don't remember who wrote this originally but I was going through some of
my old files and ran across it. Is this your work Bennet?
Does HIV Exist?
It may come as some surprise to people but there is a group of people who
maintain that HIV, a causitive agent for AIDS, does not even exist. You read
that right; there is a group headed by Eleni Papadopulos-Eleopulos, Valendar
Turner, John Papadimitriou, David Causer and Stefan Lanka, commonly referred
to as the Perth Group because they are based in Perth, Australia, who,
incredible as it may seem, claim that HIV does not even exist. They have a
web site called Virusmyth.com, (http://www.virusmyth.com) where their
arguments, such as they are, are laid out. While their views have long ago
been refuted by a mountain of evidence, they cling to their belief in the
face of overwhelming evidence that HIV doesn't exist, so cannot be the cause
of the AIDS pandemic that is now ravaging the African subcontinent. They
have even issued a $25,000 challenge to the research community to prove the
existence of the virus (http://www.virusmyth.com/aids/award.htm);
The folks at Virusmyth have established one of those contests that the
uncritical among us seem to think has some semblance of perspicacious
thought. We see these from time to time; there is Kent Hovind's $250,000
reward for "empirical evidence of evolution" (http://www.drdino.com/), for
example. Challenges such as this are not intrinsically daft, they can be
quite effective when the arguments are sound, the challenge is simple and
the conditions are not otherwise strawmen. There is, for example, James
Randi's well constructed challenge for evidence of the paranormal
(http://www.randi.org/research/challenge/index.html ). On the face of it,
the Virusmyth.com challenge seems scientifically rigorous yet simple; the
hallmarks of a good challenge. Unfortunately for the folks at Perth, it
merely seems so.
One thing is clear from the response to the researcher who's claimed the
prize; the folks over at Virusmyth.com have no intention of allowing the
contest to be won. The researcher who has claimed the prize, by the way, is
Peter Duesberg, an iconoclastic Berkeley retrovirologist who claims that
although HIV does indeed exist, it is not the cause of AIDS. The Perth Group
has taken steps to ensure that their challenge will remain unanswered. They
do this partly by setting unreasonable rules, partly by constructing
strawmen, and partly by moving the goalposts.
Unreasonable Rules
The challenge itself, is carefully crafted to ensure that no-one is likely
to take them up. This ensures that they can continue to claim that as the
challenge has not been satisfied, their arguments must be devastating to the
research and medical establishment. The reason for this is simply that the
Perth group insists that the proof for the existence of HIV can arise from
their method alone. No other evidence is acceptable. Basically they have
seven steps (below) that they claim are required to prove that HIV is real.
Imagine a reward for proof that the earth is round. But then requiring for
proof that claimants to the prize must travel to the moon and take a
picture. No other methods, from any other source at any other time would be
acceptable.
While flying to the moon and taking pictures of the earth would indeed prove
that we live on a round ball of mud, it is expensive, dangerous and wholly
unnecessary for that purpose. We could, for example, ask someone to walk
several hundred miles, look down a well and measure the angle of the sun at
noon and compare it to a well near us. Or we could sit by a bay on a calm
clear day and watch boats sail away. Or we could plot our path through the
heavens. Or we could observe the shadow of the earth ON the moon. Or we
could circumnavigate the earth in sailing ships and plot our latitude. Or we
could ascend in a balloon, or in an airplane, or launch a satellite. All of
these methods have been used, some since ancient times, to demonstrate that
the earth is round.
We do not need to isolate HIV by the techniques cited by the Perth group to
prove its existence. To insist upon their method to establish the reality of
HIV is to unequivocally demonstrate one's credulity before charlatans.
A Classic Strawman Argument
A strawman argument is an argument that uses a contrived and false premise
that is used expressly to deconstruct another argument. The people at
Virusmyth.com claim that since HIV researchers have not used guidelines
established in 1973 at the Institute Pasteur to purify HIV, then the very
existence of the virus is questionable. Unfortunately for the Perth Group,
no such guidelines were established at Pasteur or anywhere else.
Despite what it says at their site, the whole challenge is based upon the
biggest hairiest strawman I've ever seen. From the Virusmyth.com web site
(http://www.virusmyth.com/aids/award.htm);
"The rules for isolation of a retrovirus were thoroughly discussed at the
Pasteur Institute, Paris, in 1973, and are the logical minimum requirements
for establishing the independent existence of HIV. They are:
1.Culture of putatively infected tissue.
2. Purification of specimens by density gradient ultracentrifugation.
3. Electron micrographs of particles exhibiting the morfological (sic)
characteristics and dimensions (100-120nm) of retroviral particles at the
sucrose (or percoll) density of 1.16 gm/ml and containing nothing else, not
even particles of other morphologies or dimensions.
4. Proof that the particles contain reverse transcriptase.
5. Analysis of the particles' proteins and RNA and proof that these are
unique.
6. Proof that 1-5 are a property only of putatively infected tissues and can
not be induced in control cultures. These are identical cultures, that is,
tissues obtained from matched, unhealthy subjects and cultured under
identical conditions differing only in that they are not putatively infected
with a retrovirus.
7. Proof that the particles are infectious, that is when PURE particles are
introduced into an uninfected culture or animal, the identical particle is
obtained as shown by repeating steps 1-5."
Edward King (http://www.users.dircon.co.uk/~eking/index.htm) published a
rebuttal to the Virusmyth challenge in AIDS Treatment Update
(http://www.virusmyth.com/aids/news/ekisolation.htm). From that essay.
"Contrary to the implication by Continuum, the Pasteur Institute did not
draw up such guidelines in 1973. When we asked Continuum to provide the
reference for a published account of the Pasteur Institute's guidelines,
they could only supply two papers which did describe research into
retroviruses, but did not themselves meet the seven steps Continuum was now
requesting for HIV. Ironically, the authors of the papers cited by Continuum
were also the first to describe the isolation of HIV in 1983."
Indeed, those two papers cited by the Perth Group are;
Sinoussi F, Mendiola L, Chermann JC. (1973). Purification and partial
differentiation of the particles of murine sarcoma virus (M. MSV) according
to their sedimentation rates in sucrose density gradients. Spectra
4:237-243. Toplin I. (1973). Tumor Virus Purification using Zonal Rotors.
Spectra 4:225-235.
Spectra is an obscure French-Canadian journal and is blastedly hard to get
hold of. The journal is available in the U.S. only at large University
libraries with comprehensive journal collections. Still, the papers ARE
available. They DO NOT use guidelines from the Pasteur Institute. Further,
take a gander at that first author's name on the first paper cited.
Recognize her? She was a member of the group who first isolated HIV in 1983.
Her paper is cited below.
Virusmyth responds to the Dr. King's point about the absurdity of basing a
challenge to purify the virus on non-existent "guidelines" by agreeing that
the papers they site for evidence for these guidelines do not, in fact,
follow them (http://www.virusmyth.com/aids/data/epreplyek.htm). One is left
to wonder, then, why they are surprised that few people take them seriously.
Amphiboly
The dead give away for intellectual dishonesty is the practice of amphiboly,
or the use of equivocal, poorly worded or murkily-stated premises to further
an argument. Back in school when faced with an assignment from a challenging
professor that we were unable to meet, we used to refer to this strategy
this way; "if you can't dazzle them with brilliance, baffle them with
bullshit". The folks at Virusmyth use a form of amphiboly known as moving
the goalposts, wherein the premise of an argument is changed when the
argument is specifically refuted.
For example, when Ed King refuted their claim that the Pasteur Institute did
not establish the so-called guidelines for proving the existence of a
retrovirus and when Virusmyth was forced to admit that the papers they claim
supported their position did not in fact do so, they suddenly switched
tactics. They claimed that although the Spectra authors did not use the
non-existent Pasteur guidelines, they did not need to because those authors
were purifying RNA tumor viruses
(http://www.virusmyth.com/aids/data/epreplyek.htm).
When challenged by Peter Duesberg to explain why 19 full length clones of
HIV does not constitute proof that the viral genome exists, they claim that
it is because the viral genomes are not all the same size or sequence
(http://www.virusmyth.com/aids/data/epreplypd2.htm). Here they conveniently
ignore the very well known fact that RT is highly error prone. More
convenient for Virusmyth, is that by changing the subject they believe they
have rebutted Duesberg's argument.
The reader will also note the vague and undefined nature of some of their
demands in the challenge. For example they require that tissue from
"matched, unhealthy subjects" be used to isolate highly purified virions
which are then used to infect reputedly uninfected tissue. As they leave the
term "unhealthy" undefined, they retain the ability to claim that studies
which, in fact demonstrate just this, are not valid because the subjects
were not either properly matched or unhealthy
(http://www.virusmyth.com/aids/data/epcomreplypd.htm ).
(Note; you will note that the text hyperlinked in the previous sentence is
touted at the Virusmyth web site as a rebuttal to Duesberg's claim. I leave
it to the reader to decide if, in fact, Virusmyth addressed Duesberg).
Addressing The Challenge
One of the main reasons why few researchers have, or would, take the
challenge is because there simply is no percentage in it; it is clear that
any claims to the reward will be dodged, the challenge while technically
feasible is costly, and the language of the challenge is so inexact as to be
nearly meaningless. But the most important reason why few people would claim
the prize is that all the conditions to prove the existence of HIV have
already been met.
This challenge is in some ways akin to the absurd comment by Kary Mullis who
has said that HIV cannot be the cause of AIDS because there isn't one paper
that demonstrates that does
(http://www.valleyadvocate.com/hiv-ai...html#foreward). The
absurdity of this claim has been pointed out to Dr. Mullis over and over
again to no avail; the man still believes that the lack of proof for the
cause of AIDS by HIV in a single paper is sufficient for him to reject the
HIV/AIDS causality. The people at Virumyth have taken this kind of
sophomoric thinking to heart. They insist that HIV cannot be proven to even
exist unless the seven steps that they (wrongly) claim are required to prove
the existence of the virus are done in a single study. Of course all of the
steps listed by them have been done, several of them concurrently in a
single study. But they continue to insist that because no one has done the
experiments in the way they deem necessary, then the virus has not been
proven to exist.
There are literally hundreds of papers in the primary literature with
excellent images of the virus in various stages including within an infected
cell, budding from a cell or free from any cells. There are even numerous
such photos published on the web.
http://medstat.med.utah.edu/WebPath/...DS/AIDS.html#1
http://www1.omi.tulane.edu/departmen...FIGSTable.html
http://www.uni2.dk/home/inflab/
http://wwwpp.uwrf.edu/~kk00/hivvector/hivvector.html
http://www.iapac.org/clinmgt/avtherapies/saq6.html
http://www.unsw.edu.au/clients/micro...ureen/fig5.htm
http://wwwpp.uwrf.edu/~kk00/poster/HIV/HIV.htm
http://bioinformatik.biochemtech.uni...herapy/hiv.htm
http://www.cmsp.com/data2/im101.htm
http://www.sci-imagemakers.com/markus.html
http://www.lifelong.com/carnivalWorld/SEM/
http://life.anu.edu.au/viruses/ICTVdB/61065001.htm
http://telpath2.med.utah.edu/WebPath.../EM/EM017.html
http://www.avert.org/virus.htm
http://www.thebody.com/niaid/hiv_lifecycle/virbud.html
http://www.accessexcellence.org/AE/A..._Particles.GIF
http://www.cmsp.com/data2/fx100003.htm
http://www.micro.unsw.edu.au/maureen/gen-info.htm
http://www.tulane.edu/~dmsander/Big_...y/BVretro.html
Below I present some of the relevant literature that meets their demands.
Note that I give a restricted, limited citation list. In most cases there
are many more papers (and probably some that make the point better than the
ones I cite here) that could be cited but are not. I indicate the conditions
for the challenge laid forth by the Perth people with a (PP) before the
number.
(PP)1.Culture of putatively infected tissue.
This one is easy. In fact culture of "putatively" infected tissue was first
done way back in 1983 by Robert Gallo's group at the NIH in the US and Luc
Montaigner's crew at the Institute Pasteur in Paris (this is in fact, how
the virus was first identified) see; Gallo, RC et al. Science. 1983 May
20;220 (4599):865-7 and Barre-Sinoussi F, et al. Science 1983 May
20;220(4599):868-871.
Later, following the acrimony about just who isolated the first virus, the
issue was revisited. "Two of the first human immunodeficiency virus type-1
(HIV- 1) strains isolated were authenticated by reanalyzing original
cultured samples stored at the Collection Nationale de Culture des
Microorganismes as well as uncultured primary material". From; Wain- Hobson
S, et al. Science 1991 May 17;252(5008):961-5.
HIV can grow in chimpanzees (though it rarely causes disease) and in primary
cell cultures. See; Castro BA, et al. J Med Primatol 198918(3- 4):337-42
HIV-1 culture isolates were obtained from the lymph nodes and peripheral
blood mononuclear cells from 11 HIV-infected patients. See; AIDS 1994
Aug;8(8):1083-8 Tamalet C et al.
Typically, patient tissue culture isolates are derived from initial primary
cultures and clones of the virus are isolated by subsequent passage through
other cell types. See, for example; Saag MS, et al. Nature 1988 Aug 4;334
(6181):440-4, and Cheng-Mayer C, et al. Virology 1991 Mar;181(1):288-94.
Intrinsic biological properties, such as syncytia formation, cell tropism
and cytopathogenicity of different strains of HIV have been assessed by
growing primary and secondary cultures. See; von Briesen H, et al. J Med
Virol 1987 Sep;23(1):51-66.
In fact, even defective HIV, that is HIV that grows very poorly and had an
atypical Western blot and ELISA profile, has been cultured from tissue
derived from patients. See; Huet T, et al. AIDS 1989 Nov;3 (11):707-15.
There are a great many more reports of primary tissue culture isolates of
HIV. Most workers, however, use the far easier, more sensitive and cheaper
method of PCR. Even so, some researchers used tissue culture of primary HIV
isolates from patients infected with HIV to evaluate the cytopathogenicity,
cell tropism, replication capacities of different viral strains and the
correlation to clinical status. See; Lu W, Andrieu JM J Virol 1992
Jan;66(1):334-40.
(PP) 2. Purification of specimens by density gradient ultracentrifugation.
The Perth Group seems to have a fixation on this method, so let's take a
quick look at it, shall we? Density gradient ultracentrifugation is an
isopycnic method of separating thingies based on their relative densities.
Most (but not all) retroviruses have a density of 1.16 g/ml (~35% w/w
sucrose). Here's the thing; that density is NOT a unique characteristic of
HIV or even retroviruses. That is; it is an extrinsic quality. Here's an
analogy; I know that anyone who has seen the Monty Python movie the Holy
Grail (or whatever) will remember that scene where Sir Bedevere is trying to
get those English peasants to figure out what floats on water. They came up
with (I think) wood, ducks and very small rocks. Same thing here; lots of
stuff could sediment at 1.16 g/ml. In fact, everything that has a density
of..1.16 g/ml.
Nevertheless, real scientists use the technique to isolate and purify HIV.
In one paper, by Yamamoto S, et al.( J. Virol. Methods 1996 Sep; 61
(1-2):135-43) the authors used density banding to isolate viral particles
and compared the qualitative and quantitative detection of reverse
transcriptase (see below for the significance of this).
It IS true, as noted by the Perth people, that standard HIV-1 particle
preparations created with sucrose density-equilibrium gradients are
contaminated with cell-derived microvesicles, see; Bess JW Jr Virology. 1997
Mar 31;230(1):134-44 and Gluschankof P, et al. Virology 1997 Mar
31;230(1):125- 33. This does not, of course, mean that HIV banding at 1.16
g/ml is non- existent, nor does it mean that the virions cannot be separated
from the microvesicles, see; Ott DE, et al. J Virol 1996 Nov;70(11):7734-43
and, for a more recent report; Dettenhofer M, Yu XF J Virol 1999
Feb;73(2):1460-7
(PP) 3. Electron micrographs of particles exhibiting the morfological (sic)
characteristics and dimensions (100-120nm) of retroviral particles at the
sucrose (or percoll) density of 1.16 gm/ml and containing nothing else, not
even particles of other morphologies or dimensions.
Note here the devious nature of their challenge and one of the reasons why
they will never accept a claim to the reward. As noted by Edward King
"scientists have highlighted the irrelevance of this insistence on purity if
the HIV particles themselves are clearly present; for example, it's like
saying that it is impossible to identify a German Shepherd dog by its unique
appearance, if it happens to be surrounded by a pack of poodles."
But what evidence do real scientists have? Well here's a bit;
Viral particle size is usually measured by either electron microscopy (for a
direct measurement of HIV size, see; Gentile M, et al. J Virol Methods 1994
Jun;48(1):43-52, and Garnier, L, et al. J. Virol. 1999 Mar;73(3):2309-20) or
by rate zonal sedimentation (see; Garnier, L, et al J Virol 1998
Jun;72(6):4667- 77) By the way HIV, like many other retroviruses is about
80-120 nm in diameter.
"Electron microscopy of gradient-enriched preparations from supernatants of
virus-infected cells revealed an excess of vesicles with a size range of
about 50-500 nm, as opposed to a minor population of virus particles of
about 100 nm. Electron micrographs of infected cells showed polarized
vesiculation of the cell membrane, and virus budding was frequently
colocalized with nonviral membrane vesiculation." From; Gluschankof P, et
al. Virology 1997 Mar 31;230 (1):125-33. See also; Meerloo T, et al. J Gen
Virol. 1993 Jan;74:129-35.
Fortunately for the rest of the world, very few people take the Perth People
seriously. There is a great deal of effort underway to generate a vaccine.
One of the things that is likely to be required for an effective vaccine is
a highly pure, homogenous batch of HIV that is inactive (so that people do
not get infected from the vaccine). This has been accomplished, see;
Richieri SP, et al. Vaccine 1998 Jan-Feb;16 (2-3):119-29. These folks even
have very nice thin section electron microscopy evidence showing a
homogenous field of intact viral particles. They purified the HIV particles
by both anion-exchange chromatography and by sucrose density gradient
ultracentrifugation.
Some workers have even isolated viral cores. After first purifying and
concentration the virions themselves, the viral capsules are then removed by
detergent and the cores containing the viral genome and associated proteins
is visualized by EM (Welker R. et al. J Virol 2000 Feb;74(3):1168-77).
(PP)4. Proof that the particles contain reverse transcriptase.
Done. In intact virions the process is called natural endogenous reverse
transcription (NERT) and has been demonstrated, see; Zhang H, et al. J Virol
1996 May;70(5):2809-24, Zhang H, et al. AIDS Res Hum Retroviruses 1998
Apr;14 Suppl 1:S93-5, and Busso M, Resnick L J Virol Methods 1994
Apr;47(1-2):129-39 (also shown in SIV; see Dornadula G, et al. Virology
1997 Jan 6;227(1):260-7). Yamamoto S, et al.( J. Virol. Methods 1996 Sep;
61(1-2):135- 43) used density banding to isolate viral particles and
compared the qualitative and quantitative detection of reverse transcriptase
assays. In fact, one can even measure intraviral RT activity in the blood of
patients who are positive for HIV; Zhang H, et al. J Virol 1996 Jan;70
(1):628-34.
In the course of looking for a vpr gene protein in HIV, HIV particles were
banded on a sucrose density gradient and reverse transcriptase activity was
detected in just the fractions expected for a retrovirus (Cohen et al J.
Virology 64:3097-3099, 1990). RT activity can be detected in the (cell free)
sera of infected people but not in the sera of uninfected people (Heneine W
et al J Infect Dis 1995 May;171(5):1210-6, Pyra H., et al. Proc Natl Acad
Sci U S A 1994 Feb 15;91(4):1544-8, Boni J., et al. J Med Virol 1996
May;49(1):23-8
One report demonstrates that antiretroviral drugs work even on highly
purified virions. The RT activity of HIV occurs primarily in the cytoplasm
of the infected cell, but there is evidence that sometimes the virions can
initiate reverse transcription prior to infection (Lori et al. J Virol 1992
Aug;66(8):5067-74) RT inhibitors inhibited transcription of RT activity
associated with highly purified virions (see Ventura, M., et al, Arch Virol
1999;144(3):513-23
As noted above, HIV virions have even been shown to contain HIV DNA (Lori F,
et al. J Virol 1992 Aug; 66(8):5067-74). As HIV is a retrovirus, I will
leave it to the reader to consider the problem for Virusmyth in explaining
where, exactly, retroviral DNA found in the virions comes from.
(PP)5. Analysis of the particles' proteins and RNA and proof that these are
unique.
Well, apart from the RT examples above, I'll just give some of the evidence
for the viral protein, gag. There is, of course, a lot of the same evidence
available for other viral proteins, but in the interest of my nascent carpal
tunnel syndrome, I'll stick to gag.
The following papers used ultracentrifugation to purify HIV virions. There
is, of course, a huge amount of evidence based on the more powerful,
specific and sensitive PCR techniques, but I'll stick to the Perth Group's
need for this particular methodology. Evidence for sequence determinants of
HIV genome encoded gag genes that control the size, shape, morphogenesis and
budding of viral particles purified by ultracentrifugation; Garnier, L, et
al J Virol 1998 Jun;72(6):4667-77, Wang CT, et al. J Virol. 1998
Oct;72(10):7950-9, Dawson L & Yu, XF Virology 1998 Nov 10;251(1):141-57 and
Reicin AS, et al. J Virol 1996 Dec;70(12):8645-52.
(PP)6. Proof that 1-5 are a property only of putatively infected tissues and
can not be induced in control cultures. These are identical cultures, that
is, tissues obtained from matched, unhealthy subjects and cultured under
identical conditions differing only in that they are not putatively infected
with a retrovirus.
Ah, well. We now come across another canard of the Perth Group; "unhealthy
subjects". Weasel room, if I've ever seen it. You see; no matter how many
times the experiment is done, they can dodge claims to the prize by saying
something to the effect of; "ah, but since your controls did not have
(insert lacking illness here), they are not proper controls. Therefore HIV
doesn't exist."
**Sigh** What can anyone say to this? Well not much. However, controls like
this have been done since the very earliest days of the epidemic. For
example, in a very early report patients with AIDS had serum that contained
anti-HTLV antibodies while serum from 25 patients who did not have AIDS did
not react to HTLV (early on in the epidemic when it was clear that it was
caused by an infectious agent, most likely a virus and prior to the
identification of HIV, it was thought that the virus was actually HTLV).
Karpas A, et al. Mol Biol Med 1983 Nov;1(4):457-459.
See Gallo, RC et al. Science. 1983 May 20;220(4599):865-7 and Barre-
Sinoussi F, et al. Science 1983 May 20;220(4599):868-871 for the original
papers on the identification of HIV (called HTLV-III by Gallo and LAV by
Montagnier). They used non-infected tissue controls. Also; Gelmann EP, et
al. Science 1983 May 20;220(4599):862-865
(PP)7. Proof that the particles are infectious, that is when PURE particles
are introduced into an uninfected culture or animal, the identical particle
is obtained as shown by repeating steps 1-5.
Leaving aside the Perth Group's required degree of purity, infecting cells
with virus derived from infected people has been done since very early on in
the epidemic. It is a routine way to derive patient isolates or to obtain
viral clones (see, for example; Saag MS, et al. Nature 1988 Aug
4;334(6181):440- 4, and Cheng-Mayer C, et al. Virology 1991
Mar;181(1):288-94. As noted above).
13. Fisher AG, Collalti E, Ratner L, Gallo RC and Wong-Staal F: A molecular
clone of HTLV-III with biological activity. Nature (London) 316:262-265
(1985).
14. Levy JA, Cheng-Mayer C, Dina D and Luciw PA: AIDS retrovirus (ARV-2)
clone replicates in transfected human and animal fibroblasts. Science
232:998-1001 (1986).
15. Barnett SW, Quiroga M, Werner Am, Dina D and Levy JA: Distinguishing
features of an infectious molecular clone of the highly divergent and
non-cytopathic human immunodeficiency virus type 2 UC1 strain. J Virol.
67:1006-1014 (1993).
--
Gary Stein
ge.stein@verizon.net
On Sat, 19 Feb 2005 07:43:19 -0500, "PaulKing"
<aimulti@aimultimedia.com> wrote:
Then of course neither does hepatitis B or C, HPV, FIV, CAEV,
herpesviruses, or syphilis or tuberculosis or mycobacteria or
influenza.....
It has been isolated. I don't need to say anything because that's the
fact!
Nope. You're the one who is clinging desperately to the flotsam and
jetsam of your mind.
George M. Carter
**
See
http://www.miltenyibiotec.com/servic...P-Iso-HIV1.pdf
**
http://www.bioline.org.br/request?oc96135
**
Takehisa J, Zekeng L, Ido E, Yamaguchi-Kabata Y, Mboudjeka I, Harada
Y, Miura T, Kaptu L, Hayami M. Human immunodeficiency virus type 1
intergroup (M/O) recombination in cameroon. J Virol. 1999
Aug;73(8):6810-20.
Laboratory of Viral Pathogenesis, Institute for Virus Research,
Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Here we describe, for the first time, recombinants between two
highly divergent major groups of human immunodeficiency virus type 1
(HIV-1), M and O, within a Cameroonian woman infected with three
different HIV-1 strains, a group O virus, a subtype D virus, and a
recently reported IBNG (A/G)-like recombinant virus. Using nested
extra-long PCR amplification, we sequenced from the pol region to the
env region including accessory genes of the viral genome obtained from
the patient's uncultured peripheral blood mononuclear cells and
examined the phylogenetic position of each gene. Compared with
sequential blood samples obtained in 1995 and 1996, there were
multiple segmental exchanges between three HIV-1 strains (O, D, and
IBNG) and all the recombinants appeared to be derived from a common
M/O ancestor. Importantly, recombination between groups M and O
occurred, even though the homology between these two groups is 69, 76,
68, and 55% in the gag, pol, vif-vpr, and env regions, respectively.
Recombination between strains with such distant lineages may
contribute substantially to generating new HIV-1 variants.
**
Bhattacharya B, Weiss RA, Davis C, Holmes H, Hockley D, Fassati A.
Detection and quantitation of human immunodeficiency virus type-1
particles by confocal microscopy. J Virol Methods. 2004 Sep
1;120(1):13-21.
Department of Immunology and Molecular Pathology, Windeyer Institute
of Medical Sciences, Medical School, Royal Free and University College
London, 46 Cleveland Street, London W1T 4JF, UK.
A method is described to visualise directly human immunodeficiency
virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed
onto a plastic surface and doubly labeled with antibodies specific for
viral proteins and sensitive nucleic acids dyes. Laser scanning
confocal microscopy detected co-localization of viral proteins and
nucleic acids, thus allowing specific identification of HIV. Using
this technique, we have quantified eight different HIV-1 sub-types and
three HIV-1 groups in tissue culture supernatants from infected
peripheral blood mononuclear cells (PBMCs). Confocal counts correlated
well with electron microscopy (EM) counts and HIV-1 RNA loads as
determined by quantitative PCR. Confocal microscopy may prove to be a
simple alternative to electron microscopy for virus identification and
quantitation.
**
Morita T, Kawabata H, Yanagihara Y, Horikoshi Y, Mimaya J. Time-course
detection of HIV-1 proviral DNA and genomic RNA by polymerase chain
reaction in sera from seropositive and seronegative hemophiliacs
treated with clotting factor concentrates. Int J Hematol. 1993
Oct;58(3):225-32.
Laboratory of Environmental Microbiology, Graduate School of
Nutritional and Environmental Sciences, University of Shizuoka, Japan.
The detection of HIV-1 proviral DNA and genomic RNA was performed
by polymerase chain reaction (PCR) in hemophiliacs treated with
non-heated clotting factor concentrates. Reamplification with double
PCR was performed on those samples that were negative for single PCR.
Primer pairs of the gag, env, and pol regions were used for the
amplification of HIV-1 proviral DNA sequences. Amplification of the
gag region by the SK38/SK39 primer pair was useful for the detection
of proviral DNA sequences. With double PCR, 44 of 47 seropositive
samples (93.6%) were PCR-positive. All 23 seronegative samples were
PCR-negative. Reverse transcription and PCR amplification (RT-PCR)
according to the primer pair of the gag region were performed to
detect HIV-1 genomic RNA sequences. Double RT-PCR analysis of the
HIV-1 RNA sequence in frozen-preserved sera revealed that 49 of 55
seropositive sera (89.1%) were PCR-positive. Although quantification
of the PCR method was not performed in this study, we concluded that,
in patients in whom proviral DNA or genomic RNA sequences are detected
with difficulty with PCR, the onset and progression of HIV-1 infection
is delayed.
