Permission to repost.
The following is an article which Invest in ME said they would post in
their last newsletter. When the letter was released the article was not
included and they said they had forgotten to post it. Instead of
waiting for the next newsletter which could be months away, they were
asked if they would post this article on their website. Their reply was
that they would ask the person who ran the site (Richard) to do so when
he had the time. This was two months ago.
The scientific evidence for murine related retroviruses as causal in myalgic encephalomyelitis.
Introduction
Several genetically distinct murine related retroviral nucleic acid
sequences have been detected in human cells and plasma (1-3).
Transmission electron microscope imaging has demonstrated that murine
related retroviruses have type C morphology and are clearly replicating
in human cells (2). Specific immune responses have been observed in
infected people, which prove beyond reasonable doubt that the viruses
are very closely related to a family of viruses that cause cancer and
severe neurological disease in mice (2,5). Proteins produced by
integrated viral DNA have been repeatedly observed using western blot
and IHC staining techniques (4,5). These proteins have been detected
within human cells and in plasma. Nucleic acids have also been detected
within the nuclei of human cells using FISH staining techniques (5).
This nucleic acid is called copy DNA and can only enter nuclei by
forming something called a pre integrative complex. Only replicating
retroviruses can do this. Some of these sequences have been described
as belonging to xenotropic or polytropic MRVs. This indicates whether
these viruses could potentially infect mice or not.
There is considerable confusion regarding the label XMRV. This label
has been given to a particular proviral DNA sequence. Unfortunately
this sequence is highly unlikely to be related to any MRV sequences that
have been detected in humans. To avoid confusion this article will
name the MRV sequences that have been detected in the human population
as HMRV and the artificial sequence as AMRV, where A stands for
artificial. It will become readily apparent that the various tests that
have been calibrated to detect the AMRV in laboratory samples or
monkeys have been understandably unable to detect HMRV’s in infected
people.
Clinical validation
The researchers who have found HMRVs have all used techniques capable of
detecting a wide range of genetically different viral sequences (1-3)
calibrated their assays until they were able to detect proteins or
nucleic acids in people known to be infected using other approaches or
both (4,5). The ones who have failed to find any evidence of MRVs have
constructed their assays in such a way as to be only able to detect the
AMRV sequences or AMRV proteins in artificial situations (6-14). The
work of Danielson et al. is particularly intriguing (15). This team was
able to detect HMRV ENV sequences once a minimum concentration of
proviral DNA was achieved. They were however unable to detect GAG or
POL sequences. The technique used did allow for the detection of a
little sequence variation compared to the AMRV sequence but still could
not detect GAG or POL sequences. It could simply mean that the GAG and
POL sequences were not present or not accessible for some reason. It
could also mean that the GAG or POL sequences were so different to those
of the AMRV that the assay was not capable of detecting them. In any
event the requirement of a minimal concentration of DNA alone would
explain the findings of Groom (10) and Erlwein (7,8) who used
comparatively tiny amounts of DNA.
What other reasons account for the failure of PCR?
Even if a HMRV is genetically very similar to the AMRV there are a
number of reasons why a PCR assay would fail to detect it in an infected
person. The two most common reasons for PCR failure are low copy
number of target sequence and the presence of secondary structures. The
first is obvious, but perhaps the relevance of the second factor when
it comes to HMRV detection is less so. In essence HMRVs are very likely
to be integrated into the host genome into promoter regions (CpG
islands) of genes and may well not express proteins while in this latent
state (16-18). These CpG islands are very difficult to amplify by PCR
and require modifications in annealing temperature and time as well as
magnesium concentration, which is not needed to detect AMRV DNA in a
spiked sample in vitro (19, 20). Hence without adjusting PCR conditions
used to detect the AMRV DNA in vitro, the assay could fail to detect a
HMRV in an infected person. The presence of secondary structures in
such areas could well render the target sequence invisible to such a
number of primers that the amplification of a low copy integrated virus
could simply fail in the amplification of DNA.
Oxidative stress in neurological diseases and how it affects PCR.
Patients with ME, in common with other neuroimmune disease, have high
levels of oxidative stress. This can lead to oxidatively damaged DNA
(21). Oxidative damage to DNA is a very common reason for PCR failure
(22,23).
This would be a very simple explanation for the failure of PCR assays
optimized to amplify normal undamaged DNA when applied to the task of
amplifying a HMRV at low copy number integrated into oxidatively damaged
DNA. Oxidatively modified viral proteins would also explain the
failure of various serology assays to detect a HMRV.
Gamma retroviruses and their relationshiop to blood and tissue
The most parsimonious explanation for the failure to detect a HMRV in
blood is that the blood is not the natural “home” for HMRVs. The work
of Onlamoon et al. (24) and others is especially instructive in this
matter. They found that the AMRV inoculated into macaques soon became
undetectable in the blood by PCR but readily detectable by the same
methodology in tissues. The antibody response also faded rapidly. This
is typical of the behavior of a very closely related gammaretrovirus
that is the causative agent of murine Aids. This virus infects and
destroys the cells that are needed to maintain an immune response to the
virus (25). LP-bm5 def (as the virus is named) infects, activates and
resides within plasma B cells (26). These cells in the absence of an
infection are rarely if ever in the blood and hence PCR on blood
components or plasma would be very unlikely to detect this virus without
some method of activating to increase the numbers of these cells in
this compartment.
Sequence variation
Some groups have claimed that the lack of sequence variation in some
HMRVs indicates that they are contaminants (27). The alternative
explanation is that they replicate not like HIV but like delta
retroviruses (28), beta retroviruses (29) or another gammaretrovirus
LP-BM5 def (30).
Does the integration site of an MRV indicate contamination?
At least one HMRV has been detected integrated into host DNA (16, 17).
A recent study claimed that these integration sites were artifacts
(31). The basis of this claim was the fact that the infectious AMRV
clone was shown to integrate into precisely the same nucleotide in a
cell line as it did in host DNA. According to the authors no retrovirus
in history had ever demonstrated this property. This claim has been
proved to be incorrect as two other closely related gamma retroviruses
(32,33) and HTLV-1 (34) have demonstrated this property. Thus the
evidence relating to HMRV integration proving that at least one is a
replicating infectious retrovirus is robust and untarnished. Indeed in a
recent report Dr P Chaney suggests that second generation sequencing
studies on ME patients in Belgium and Germany are detecting integrated
HMRVs at levels considerably higher than in healthy controls (35).
Future progress
A vehement critic of HMRV research was recently quoted as saying that:
“…the science is done…” (36).
I would argue that no real science has yet been done. Hopefully we are
done with biopolitical studies and we can have research based on natural
science instead. I would conclude with the words of Robert Persig:
"An experiment is a failure only when it also fails adequately to test
the hypothesis in question, when the data it produces don't prove
anything one way or another.” (37)
It is time to end the publication of paper after paper whose
experimental design is such that the conclusions merely reflect the
philosophical disposition of the authors and fail to provide any
scientifically robust evidence regarding the presence or absence of
HMRVs in the populations studied. It is time for serious scientists to
take the scientific mode of investigation seriously.
Rituximab
Rituximab has several effects on the immune system, which may or may not
be related to B cell depletion. One of the most potentially surprising
effects is the reduction of Th17 T cell production (38,39). These T
cells are the cause of T cell induced autoimmunity and neurotoxicity in
other autoimmune diseases like MS. The cytokine and chemokine profile
detected in HMRV positive people is consistent with an activated but
TH17 biased immune system (40). Rituximab has a direct effect on
reducing IL-2 levels and thus potentially inactivating a chronically
activated immune system (41). Rituximab achieves this by inhibiting the
production of NF-kappa B (42-45), which is another key mediator of
autoimmunity. Rituximab also raises the function of regulatory T cells
(46,47). Reduced T reg function is also another major cause of
autoimmunity and neurotoxicity. The benefit induced by a CD20
monoclonal antibody is entirely consistent with a disease produced by a
gammaretroviral infection.
The closely related gammaretrovirus that cause murine aids (MAIDS) could
be an appropriate explanatory model. This virus inserts into the DNA
of infected B cells and stimulates the genes causing an increase in
mitosis (48). The defective GAG protein encoded by this virus causes
havoc with the host’s immune system and induces severe autoimmune
disease (49). Pathogenesis is caused by the presence of multiple
autoantibodies (50), some of which induce serious and often fatal
neurotoxicity (51). This virus is known to infect plasma B cells and
possibly pre B cells (52). It causes pathology by altering B cell
signaling via a complex mechanism involving biochemical pathways p38
MAPK, ERK and others leading to pronounced immune dysregulation and
neuroinflammation (53). Rituximab blocks B cell signalling (54) thus
the benefits conveyed by Rituximab are entirely consistent with the
disease being caused by HMRVs.
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