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.
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
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
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
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).
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 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
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.
1. Lo et al. Detection of MLV-related virus gene sequences in blood
of patients with chronic fatigue syndrome and healthy blood donors.
Proc Natl Acad Sci. 2010. doi: 10.1073/pnas.1006901107 http://www.pnas.org/content/early/2010/0...7.abstract
2. Lombardi et al. Detection of an infectious retrovirus, XMRV, in
blood cells of patients with chronic fatigue syndrome. Science. 2009.
3. Switzer et al. No association of xenotropic murine leukemia
virus-related viruses with prostate cancer. PLoS One. 2011.
4. Schlaberg et al. XMRV is present in malignant prostatic
epithelium and is associated with prostate cancer, especially high-grade
tumors. Proc Natl Acad Sci. 2009. doi: 10.1073/pnas.0906922106 http://www.pnas.org/content/early/2009/09/04/0906922106
5. Urisman et al. Identification of a Novel Gammaretrovirus in
Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant. PLoS
Pathogens. 2006. doi:10.1371/journal.ppat.0020025 http://www.plospathogens.org/article/inf...at.0020025
6. Switzer et al. Absence of evidence of Xenotropic Murine Leukemia
Virus-related virus infection in persons with Chronic Fatigue Syndrome
and healthy controls in the United States. Retrovirology. 2010.
7. Erlwein et al. Failure to Detect the Novel Retrovirus XMRV in
Chronic Fatigue Syndrome. PLoS One. 2010.
8. Erlwein et al. Investigation into the Presence of and
Serological Response to XMRV in CFS Patients. PLoS One. 2011.
9. Robinson et al. Xenotropic murine leukaemia virus-related virus
(XMRV) does not cause chronic fatigue. Trends in Microbiology. 2011.
10. Groom et al. Absence of xenotropic murine leukaemia virus-related
virus in UK patients with chronic fatigue syndrome. Retrovirology. 2010.
11. van Kuppeveld et al. Prevalence of xenotropic murine leukaemia
virus-related virus in patients with chronic fatigue syndrome in the
Netherlands: retrospective analysis of samples from an established
cohort. BMJ. 2010. doi:10.1136/bmj.c1018 http://www.bmj.com/content/340/bmj.c1018.full
12. Knox et al. No Evidence of Murine-Like Gammaretroviruses in CFS
Patients Previously Identified as XMRV-Infected. Science. 2011. DOI:
13. Shin et al. Absence of XMRV and other MLV-related viruses in
patients with Chronic Fatigue Syndrome. Journal of Virology. 2011.
doi:10.1128/ JVI.00693-11 http://jvi.asm.org/content/early/2011/05...1.abstract
14. Elfaitouri et al. Murine Gammaretrovirus Group G3 Was Not Found in
Swedish Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome
and Fibromyalgia. PLoS One. 2011. doi:10.1371/journal.pone.0024602 http://www.plosone.org/article/info%3Ado...ne.0024602
15. Danielson et al. Detection of Xenotropic Murine Leukemia Virus–
Related Virus in Normal and Tumor Tissue of Patients from the Southern
United States with Prostate Cancer Is Dependent on Specific Polymerase
Chain Reaction Conditions. J of Infectious Diseases. 2010. DOI:
16. Dong et al. An infectious retrovirus susceptible to an IFN
antiviral pathway from human prostate tumors. Proc Natl Acad Sci. 2007.
doi: 10.1073/pnas.0610291104 http://www.pnas.org/content/104/5/1655
17. Kim et al. Integration site preference of xenotropic murine
leukemia virus-related virus, a new human retrovirus associated with
prostate cancer. J Virol. 2008. doi: 10.1128/ JVI.01299-08 http://jvi.asm.org/content/82/20/9964.short
18. Kim et al. Fidelity of target site duplication and sequence
preference during integration of xenotropic murine leukemia
virus-related virus. PLoS One. 2010. doi:10.1371/journal.pone.0010255 http://www.plosone.org/article/info%3Ado...ne.0010255
19. Saxonov et al. A genome-wide analysis of CpG dinucleotides in the
human genome distinguishes two distinct classes of promoters. Proc Natl
Acad Sci. 2006. doi:10.1073/pnas.0510310103 http://www.pnas.org/content/103/5/1412.full
20. Sol-Church et al. Pyrosequencing submission guidelines. Nemours. 2002. http://medsci.udel.edu/cores/bcl/forms/P...elines.pdf
21. Kadioglu et al. Detection of oxidative DNA damage in lymphocytes of
patients with Alzheimer's disease. Biomarkers. 2004.
22. Sikorsky et al. DNA damage reduces Taq DNA polymerase fidelity and
PCR amplification efficiency. Biochem Biophys Res Commun. 2007. doi:
23. Kathe et al. Single-Stranded Breaks in DNA but Not Oxidative DNA
Base Damages Block Transcriptional Elongation by RNA Polymerase II in
HeLa Cell Nuclear Extracts. J Biol Chem. 2004.
24. Onlamoon et al. Infection, viral dissemination, and antibody
responses of rhesus macaques exposed to the human gammaretrovirus XMRV. J
Virol. 2011. doi: 10.1128/ JVI.02411-10 http://jvi.asm.org/content/early/2011/02...0.abstract
25. Masuda et al. Loss of follicular dendritic cells in murine-acquired immunodeficiency syndrome. Lab Invest. 1995. http://www.ncbi.nlm.nih.gov/pubmed/7474923
26. Liberatore et al. Tetherin is a key effector of the antiretroviral
activity of type I interferon in vitro and in vivo. Proc Natl Acad Sci.
2011, doi: 10.1073/pnas.1113694108. http://www.pnas.org/content/early/2011/1...8.abstract
27. Garson et al. Analysis of XMRV integration sites from human
prostate cancer tissues suggests PCR contamination rather than genuine
human infection. Retrovirology. 2011. doi:10.1186/1742-4690-8-13 http://www.retrovirology.com/content/8/1/13
28. Tanaka et al. The clonal expansion of human T lymphotropic virus
type 1-infected T cells: a comparison between seroconverters and
long-term carriers. J Infect Dis. 2005. doi:10.1086/428625 http://jid.oxfordjournals.org/content/191/7/1140.full
29. Callahan et al. MMTV-induced mammary tumorigenesis: gene discovery,
progression to malignancy and cellular pathways. Ongogene. 2000. http://www.nature.com/onc/journal/v19/n8...3276a.html
30. Hitoshi et al. Delayed Progression of a Murine Retrovirus-induced
Acquired Immunodeficiency Syndrome in X-llnked Immunodeficient Mice. J.
Exp. Med. 1993. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2190950/
31. Hue et al. Disease-associated XMRV sequences are consistent with
laboratory contamination. Retrovirology. 2011.
32. Weinstein et al. Insertion and truncation of c-myb by murine
leukemia virus in a myeloid cell line derived from cultures of normal
hematopoietic cells. J Virol. 1987. http://jvi.asm.org/content/61/7/2339.abstract
33. Selten et al. The primary structure of the putative oncogene pim-1
shows extensive homology with protein kinases. Cell. 1986.
34. Meekings et al. HTLV-1 Integration into Transcriptionally Active
Genomic Regions Is Associated with Proviral Expression and with HAM/TSP.
PLoS Pathogens. 2008. doi:10.1371/journal.ppat.1000027 http://www.plospathogens.org/article/inf...at.1000027
35. Cheney P. Changing status of XMRV / HGRV research. The Cheney Clinic. 2011. http://www.cheneyclinic.com/changing-sta...arch-2/843
36. Steven Salzberg. Chronic fatigue syndrome researcher arrested. Medpedia. 2011. http://www.medpedia.com/news_analysis/6-...r-arrested
37. Robert M. Pirsig, Zen and the Art of Motorcycle Maintenance: An
Inquiry Into Values. William Morrow & Company. 1974. ISBN
38. Ireland et al. Potential Impact of B Cells on T Cell Function in
Multiple Sclerosis. Mult Scler Int. 2011. doi:10.1155/2011/423971 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197079/
39. van de Veerdonk et al. The anti-CD20 antibody rituximab reduces the
Th17 cell response. Arthritis Rheum. 2011. doi:10.1002/art.30314. http://www.ncbi.nlm.nih.gov/pubmed/21400475
40. Lombardi et al. Xenotropic Murine Leukemia Virus-related
Virus-associated Chronic Fatigue Syndrome Reveals a Distinct
Inflammatory Signature. In Vivo. 2011. http://iv.iiarjournals.org/content/25/3/307.abstract
41. Lederer et al. Regulation of NF-kappa B activation in T helper 1 and T helper 2 cells. J of Immunology. 1996. http://www.jimmunol.org/content/156/1/56.abstract
42. Jazirehi et al. Rituximab (Chimeric Anti-CD20 Monoclonal Antibody)
Inhibits the Constitutive Nuclear Factor - KB Signaling Pathway in
Non-Hodgkin’s Lymphoma B-Cell Lines: Role in Sensitization to
Chemotherapeutic Drug-induced Apoptosis. Cancer Res. 2005. http://faculty.bioscience.ucla.edu/insti..._id=122789
43. Bonavida B. Rituximab-induced inhibition of antiapoptotic cell
survival pathways: implications in chemo/immunoresistance, rituximab
unresponsiveness, prognostic and novel therapeutic interventions.
Oncogene. 2007. doi:10.1038/sj.onc.1210365 http://www.nature.com/onc/journal/v26/n2...0365a.html
44. Bonavida et al. Rituximab-mediated chemosensitization of AIDS and
non-AIDS non-Hodgkin's lymphoma. Drug Resist Updat. 2005. http://www.journals.elsevierhealth.com/p...8/abstract
45. Bonavida B. ‘Rituximab-induced inhibition of antiapoptotic cell
survival pathways: implications in chemo/immunoresistance, rituximab
unresponsiveness, prognostic and novel therapeutic
interventions’Rituximab-induced inhibition. Oncogene. 2007.
46. Vigna-Perez et al. Clinical and immunological effects of Rituximab
in patients with lupus nephritis refractory to conventional therapy: a
pilot study. Arthritis Res Ther. 2006 http://arthritis-research.com/content/8/3/R83
47. Vigna-Pérez et al. In vivo effect of Rituximab on regulatory T
cells and apoptosis in patients with rheumatoid arthritis. Inmunología.
48. Huang et al. The murine AIDS defective provirus acts as an
insertional mutagen in its infected target B cells. J of Virology. 1995.
49. Morse et al. Immunologic havoc induced by the 60 kD Gag protein of the MAIDS defective virus. Leukemia. 1997. http://www.ncbi.nlm.nih.gov/pubmed/9209332
50. Mittleman et al. Amelioration of experimental systemic lupus
erythematosus (SLE) by retrovirus infection. J Clin Immunol. 1996. http://www.ncbi.nlm.nih.gov/pubmed/8840225
51. Kousteva et al. LP-BM5 virus–infected mice produce activating
autoantibodies to the AMPA receptor. J Clin Invest. 2001.
52. Klein et al. Establishment of MAIDS-defective virus-infected B cell lines and their characterization. Virology. 1998. http://www.ncbi.nlm.nih.gov/pubmed/9601499
53. Sonja et al. CD19 Signaling Pathways Play a Major Role for Murine AIDS Induction and Progression. J of Immunology. 2002. http://www.jimmunol.org/content/169/10/5607.full.pdf
54. Kheirallah et al. Rituximab inhibits B-cell receptor signaling. Blood. 2010. doi: 10.1182/blood-2009-08-237537 http://bloodjournal.hematologylibrary.or...5/985.long