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WPI Finds High Levels of Retrovirus in ME/CFS Patients

Discussion in 'XMRV Research and Replication Studies' started by Cort, Oct 8, 2009.

  1. dkesh

    dkesh

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    My father too has both CFS and prostate cancer (fully treated). I contacted WPI and asked them if they'd be interested in researching him; I'll post here if they reply.
  2. Warbler512

    Warbler512

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    Wetting his pants?

    I wish, but not yet. People like Reeves and Wesseley didn't get to be who they are without thick skin to hold the melting icewater in their veins.

    The WPI site says "bodily fluids." To me, that includes saliva, unless someone has said otherwise, in which case it makes XMRV that much more interesting than just a ratty coincidence. WPI apparently has also come up with a new name - "XAND" (XMRV Associated Neuro. Diseases) that encompasses "CFS." We'll see about it's staying power. Nothing up yet on how we can donate our "precious bodily fluids."

    Where's Dr. Strangelove when you need him. Oh, that's Dr. Reeves. And whatever happened to the person in Osler's Web who had that letter from Reeves saying not to donate his blood? Hmmmm??????
  3. _Kim_

    _Kim_ Guest

    Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic

    Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we
    identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV),
    in 68 of 101 patients (67%) compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cellassociated and cell-free transmission of the virus are
    possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines
    following exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.


    Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we
    identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV),
    in 68 of 101 patients (67%) compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cellassociated and cell-free transmission of the virus are
    possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines
    following exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS. Chronic fatigue syndrome (CFS) is a disorder of unknown etiology that affects multiple organ systems in the body.
    Patients with CFS display abnormalties in immune system function, often including chronic activation of the innate immune system and a deficiency in natural killer (NK) cell activity (1, 2). A number of viruses, including ubiquitous herpesviruses and enteroviruses have been implicated as possible environmental triggers of CFS (1). Patients with CFS often have active  herpesvirus infections, suggesting an underlying immune deficiency.

    The recent discovery of a gammaretrovirus, XMRV, in the tumor tissue of a subset of prostate cancer patients prompted us to test whether XMRV might be associated with CFS. Both of these disorders, XMRV-positive prostate cancer and CFS, have been linked to alterations in the antiviral enzyme RNase L (3–5). Using the Whittemore Peterson Institute’s (WPI) national tissue repository, which contains samples from well-characterized cohorts of CFS, we isolated nucleic acids from PBMCs and assayed the samples for XMRV gag sequences by nested PCR (5, 6). Of the 101 CFS samples analyzed, 68 (67%) contained XMRV gag sequence. Detection of XMRV was confirmed in 7 of 11 WPI CFS
    samples at the Cleveland Clinic by PCR-amplifying and sequencing segments of XMRV env (352 nt) and gag (736 nt) in CFS PBMC DNA (Fig. 1A) (6). In contrast, XMRV gag sequences were detected in 8 of 218 (3.7%) PBMC DNA specimens from healthy individuals. Of the 11 healthy control DNA samples analyzed by PCR for both env and gag, only one sample was positive for gag and none for env (Fig. 1B). In all positive cases, the XMRV gag and env sequences were more than 99% similar to those previously reported for prostate tumor-associated strains of XMRV (VP62, VP35, and VP42) (fig. S1) (5).

    Sequences of full-length XMRV genomes from two CFS patients and a partial genome from a third patient were generated (table S1). CFS XMRV strains 1106 and 1178 each differed by six nucleotides (nt) from the reference prostate cancer strain XMRV VP62 (EF185282), and with the exception of one nt, the variant nucleotides mapped to different locations within the XMRV genome, suggesting independent infections. By comparison, prostate cancer derived XMRV strains VP35 and VP42 differed from VP62 by 13 and 10 nt, respectively. Thus, the complete XMRV genomes in CFS patients are > 99% identical in sequence to those detected in patients with prostate cancer. To exclude the possibility that we were detecting a murine leukemia virus (MLV) laboratory contaminant, we determined the phylogenetic relationship between endogenous (nonecotropic) MLV sequences, XMRV sequences, and sequences from CFS patients 1104, 1106 and 1178 (fig. S2). XMRV sequences from the CFS patients clustered with the XMRV sequences from prostate cancer cases and formed a distinct branch from nonecotropic MLVs common in inbred mouse strains. Thus, the virus detected in the CFS patients’ blood samples is unlikely to be a contaminant.

    To determine whether XMRV proteins were expressed in PBMCs from CFS patients, we developed intracellular flow cytometry (IFC) and Western blot (WB) assays, using antibodies (Abs) with novel viral specificities. These antibodies included among others: (i) rat monoclonal antibody (mAb) to the spleen focus-forming virus (SFFV) envelope (Env), which reacts with all polytropic and xenotropic MLVs (7), (ii) goat antisera to whole mouse NZB xenotropic MLV; and (iii) a rat mAb to MLV p30 Gag (8). All of these Abs detected the human VP62 XMRV strain grown in human Raji, LNCaP and Sup-T1 cells (fig. S3) (5). IFC of activated lymphocytes (6, 9) revealed that 19 of 30 PBMC samples from CFS patients reacted with the anti-MLV p30 Gag mAb (Fig. 2A). The majority of the 19 positive samples also reacted with antisera to other purified MLV proteins (fig. S4A). In contrast, 16 healthy control PBMC cultures tested negative (Fig. 2A, fig. S4A). These results were confirmed by Western blots (Fig. 2B and C) (6) using Abs to SFFV Env, mouse xenotropic MLV and MLV p30 Gag. Samples from five healthy donors exhibited no expression of XMRV proteins (Fig. 2C). The frequencies of CFS cases vs. healthy controls that were positive and negative for XMRV sequences were used to calculate a Pearson 2 value of 154 (two-tailed P value of 8.1  10–35). These data yield an odds ratio of 54.1 (95% confidence interval of 23.8-122), suggesting a non-random association with XMRV and CFS patients.

    To determine which types of lymphocytes in blood express XMRV, we isolated B and T cells from one patient’s PBMCs (6). Using mAb to MLV p30 Gag and IFC, we found that both activated T and B cells were infected with XMRV (Fig. 2D, fig. S4A). Furthermore, using mAb to SFFV Env, we found that > 95% of the cells in a B-cell line developed from another patient were positive for XMRV Env (Fig. S4B). XMRV protein expression in CFS patient-derived activated T and B cells grown for 42 days in culture was confirmed by Western blots (fig. S4C) using Abs to SFFV Env and xenotropic MLV.

    We next investigated whether the viral proteins detected in PBMCs from CFS patients represent infectious XMRV. Activated lymphocytes (6) were co-cultured with LNCaP, a prostate cancer cell line with defects in both the JAK-STAT and the RNase L pathways (10, 11) that was previously shown to be permissive for XMRV infection (12). After coculture with activated PBMCs from CFS patients, LNCaP cells expressed XMRV Env and multiple XMRV Gag proteins by Western blot (Fig. 3A) and IFC (fig. S5A). Transmission electron microscopy (EM) of the infected LNCaP cells (Fig. 3B) as well as virus preparations from these cells (Fig. 3C) revealed 90-100 nm diameter budding particles consistent with a gamma (type C) retrovirus (13).

    We also found that XMRV could be transmitted from CFS patient plasma to LNCaP cells when we applied a virus centrifugation protocol to enhance infectivity (6, 14, 15). Both XMRV gp70 Env and p30 Gag were abundantly expressed in LNCaP cells incubated with plasma samples from 10 of 12 CFS patients, whereas no viral protein expression was detected in LNCaP cells incubated with plasma samples from 12 healthy donors (Fig. 3A). Likewise, LNCaP cells incubated with patient plasma tested positive for XMRV p30 Gag in IFC assays (fig. S5B). We also observed cell-free transmission of XMRV from the PBMCs of CFS patients to the T-cell line SupT1 (Fig. 4B) and both primary and secondary transmission of cell-free virus from the activated T cells of CFS patients to normal T cell cultures (Fig.4C). Together, these results suggest that both cellassociated and cell-free transmission of CFS-associated XMRV are possible.

    We next investigated whether XMRV stimulates an immune response in CFS patients. For this purpose, we developed a flow cytometry assay that allowed us to detect antibodies to XMRV Env by exploiting its close homology to SFFV Env (16). Plasma from 9 out of 18 CFS patients infected with XMRV reacted with a mouse B cell line expressing recombinant SFFV Env (BaF3ER-SFFV-Env) but not to SFFV Env negative control cells (BaF3ER), analogous to the binding of the SFFV Env mAb to these cells (Fig. 4D and S6A). In contrast, plasma from seven healthy donors did not react (Fig. 4D and fig. S6A). Furthermore, all nine positive plasma samples from CFS patients but none of the plasma samples from healthy donors blocked the binding of the SFFV Env mAb to SFFV Env on the cell surface (fig. S6B). These results are consistent with the hypothesis that CFS patients mount a specific immune response to XMRV.

    Neurological maladies and immune dysfunction with inflammatory cytokine and chemokine upregulation are some of the most commonly reported features associated with CFS. Several retroviruses, including the MLVs and the primate retroviruses, HIV and HTLV-1, are associated with neurological diseases as well as cancer (17). Studies of retrovirus-induced neurodegeneration in rodent models have indicated that vascular and inflammatory changes mediated by cytokines and chemokines precedes the neurological pathology (18, 19). The presence of infectious XMRV in lymphocytes may account for some of these observations of altered immune responsiveness and neurological function in CFS patients.

    In summary, we have discovered a highly significant association between the XMRV retrovirus and CFS. This observation raises several important questions. Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population? What is the relationship between XMRV infection status and the presence or absence of other viruses that are often associated with CFS (e.g., herpesviruses)?

    Conceivably these viruses could be cofactors in pathogenesis, as is the case for HIV-mediated disease, where co-infecting pathogens play an important role (20). Patients with CFS have an elevated incidence of cancer (21). Does XMRV infection alter the risk of cancer development in CFS? As noted above, XMRV has been detected in prostate tumors from patients expressing a specific genetic variant of the RNASEL gene (5). In contrast, in our study of this CFS cohort, we found that XMRV infection status does not correlate with the RNASEL genotype (6) (table S2).

    Finally, it is worth noting that 3.7% of the healthy donors in our study tested positive for XMRV sequences. This suggests that several million Americans may be infected with a retrovirus of as yet unknown pathogenic potential.
  4. Finch

    Finch Down With the Sickness

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    Breathtaking!

    This is all rather breathtaking. I got the news at work this afternoon in a notice from the CFIDS Association of America. It's slowly starting to sink in. We're going to be big news, and we can only wait now to see how it all plays out.

    Definitely, something is happening! I'll sit back and wait to hear whether anyone I know brings it up to me. I told my husband, and he asked, "So what does this mean for CFS people?" I told him we don't know yet, but we'll find out!

    I'm so hoping this all pans out not only to make us more legitimate, but to help us all to find better health in the long run. Maybe even prevent the cancer we may have been destined to eventually develop otherwise. But I'm thinking way too prematurely there.

    Big, big, big news.
  5. Cort

    Cort Phoenix Rising Founder

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    They suggesting that could be passed in mothers milk; I suppose that it's possible that Andrea Whittemore got it from Annette Whittemore (who could have gotten it from Harvey Whittemore) - who knows? The transmission question is clearly going to be a central issue.

    I was a pretty low risk for any kind of transmission along those lines when I got ME/CFS. Maybe its something that hides out for years until something lets it loose. Remember they found the virus in almost 4% of healthy controls.

    I asked for an interview - we'll see!
  6. August59

    August59 Daughters High School Graduation

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    I wonder what this does to the status of Ampligen. More test, dead duck or does it give it more legs to stand on?
  7. _Kim_

    _Kim_ Guest

    And the disclaimer...

    The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
  8. cfs since 1998

    cfs since 1998 *****

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    So we have a retrovirus associated with CFS that infects at a minimum T cells and B cells, while two viruses that are often associated with CFS, Epstein-Barr and HHV-6, infect B cells and T cells, respectively. Interesting.
  9. fairlight

    fairlight

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    I think this helps Ampligen

    as Ampligen is an immune modulator. Hemisperix BioPharma (maker of Ampligen) was up more than 10% today after the news in after hours.

    Below is an exerpt from this article:
    http://www.xe.com/news/2009-10-08 14:15:00.0/725161.htm?c=1&t=

    Much more study would be necessary to show a direct link, but Mikovits said the study offers hope that CFS sufferers might gain relief from a cocktail of drugs designed to fight AIDS, cancer and inflammation

    'You can imagine a number of combination therapies that could be quite effective and could at least be used in clinical trials right away,' Mikovits said in a telephone interview.

    She said AIDS drugs such as non-nucleoside reverse transcriptase inhibitors and integrase inhibitors as well as nonsteroidal anti-inflammatory drugs and cancer-fighting proteasome inhibitors could be tested as potential treatments for CFS.

    Takeda Pharmaceutical Co Ltd makes a cancer drug called Velcade that is a proteasome inhibitor, although there are no reports that it has been tested against XMRV.
  10. acer2000

    acer2000 Senior Member

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    Interesting they mention it infects "T and B" cells, what about CD56 NK cells? You know, the ones they always say are low in CFS patients?
  11. _Kim_

    _Kim_ Guest

    Perspectives from Coffin and Stoye

    "The form of XMRV now seen in humans can be traced back to a mouse virus, the xenotropic murine leukemia virus (MLV), explain Coffin and Stoye. This is called an endogenous virus because it infects reproductive cells and can therefore be passed on to offspring of infected parents."

    Could be that some of us were born with the virus and some of us had transmission by bodily fluids.
  12. Rrrr

    Rrrr Senior Member

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    antiretroviral supplements

    a friend sent me this below info on antiretroviral supplements, if anyone is thinking of going that route (this info is not from me, i'm just forwarding the info along):

    Jon Kaiser, a very well-known AIDS doctor who has written about natural treatments for AIDS, [used the same supplements] for his CFIDS as well as AIDS patients (he gave them a similar protocol). There was this one product that was particularly good and actually helped [me]:

    <http://www.amazon.com/dp/B000OO5B5G/ref=asc_df_B000OO5B5G930342?smid>

    if we have a retrovirus like HIV, it might be worth checking out.

    I also read that Oyster and True Tinder Polypore mushrooms seem to have some efficacy against HIV. Also Self-heal (also known as Prunella), coptis (also good for bartonella), hyssop.*

    -- again, i'm just forwarding the info along --
  13. dannybex

    dannybex Senior Member

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    I don't know if this has been posted yet...

    ...but this article from the NYTimes helps explain a little more the difference in the test results:

    "An article published online Thursday in the journal Science reports that 68 of 101 patients with the syndrome, or 67 percent, were infected with an infectious virus, xenotropic murine leukemia virus-related virus, or XMRV. By contrast, only 3.7 percent of 218 healthy people were infected. Continuing work after the paper was published has found the virus in nearly 98 percent of about 300 patients with the syndrome, said Dr. Judy A. Mikovits, the lead author of the paper."

    But then again, at the same time, why was it found in 67% of the first sample, then later 98% of the latter sample?

    Okay, I'll stop asking the same question! :)

    d.

    Here's the link:

    http://www.nytimes.com/2009/10/09/health/research/09virus.html
  14. Rrrr

    Rrrr Senior Member

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    Kp-1461

    again, i'm forwarding this from a friend...

    This is really bizarre and interesting. Apparently, they are on the forefront of approving a new type of retroviral drug that causes the retrovirus to literally "mutate itself to death." HIV apparently mutates so much that it creates what are called "quasispecies" and thus the body is filled with millions of mini species that are hard to eradicate. There is a new drug in testing now called KP-1461 that, instead of killing the virus, actually tries to push it until it mutates into a catastrophic end, and then is literally cleared from the system. In other words, if it works the way it is theorized to work, one wouldn't have to take it for life but only long enough to mutate the retrovirus to an "error catastrophe" when the virus is finally and ultimately cleared from the cells. Here is an explanation since it's hard to explain this, but it's really interesting, esp. since it seems like something that could apply to other retroviruses:

    from: <http://www.aids.org/atn/2007/10/kp-1461.html>

    Summary: KP-1461, an experimental HIV drug already in a phase II trial, works so differently from other antiretrovirals that at first glance it looked like science fiction, and we found it hard to take seriously as a current possibility today. In fact this drug is highly credible, and based on elegant science that goes back at least 25 years. KP-1461 is the only antiretroviral in human use or testing that can eradicate HIV from laboratory cell cultures. No one knows how it will work in people -- but we might know by the second quarter of 2008, when the current phase II trial could be complete. AIDS Treatment News interviewed Dr. Stephen Becker, a leading AIDS physician and researcher who is vice president of clinical development at Koronis Pharmaceuticals, in Seattle, Washington.

    Background: The scientific story began when Mansfeld Eigen (who had already won a Nobel Prize in chemistry for other work) applied his chemistry and mathematics background to problems in biology, and with Peter Schuster and others developed the concept of quasispecies. Standard Darwinian evolution predicts that the fittest strain of an organism, the one that reproduces fastest in a given environment, will displace the other strains there. But a virus like HIV is different; it is always mutating, and can mutate back and forth between different strains. The result is that HIV, in a patient with advanced infection or AIDS, exists as millions of related strains within the same patient (usually only one was transmitted, and then it evolved within that individual into countless slightly different variants).

    This makes HIV hard to treat, because some members of the quasispecies probably already have resistance mutations to a new drug even by chance alone, and these resistant viruses are ready to be selected and become much more prevalent when the drug is started. The conventional approach to this problem is to use combinations of different drugs, hoping to suppress HIV to such a low level that little mutation and evolution can take place. This may suppress the virus for years, but has never succeeded in eradicating it, so patients usually have to stay on treatment for life.

    Quasispecies follow different rules than Darwinian evolution. For example, it is possible at least in theory for the strain that reproduces fastest to be replaced entirely by strains that individually reproduce more slowly, but are more fit as a quasispecies. Eigen and Schuster also wrote a well-known book, The Hypercycle: A Principle of Natural Self-Organization, published in 1979 on quasispecies and related concepts.

    A way to attack a quasispecies as a whole is to increase the already-high mutation rate, leading to an "error catastrophe" and collapse of the population. This approach was used to design the drug now in a phase II trial, KP-1461. KP-1461 is a nucleoside analog, like AZT, 3TC, and the others; once inside the cell it is chemically modified (triphosphorylated) into its active form (called KP-1212), which can replace one of the four bases used to make DNA. (The four bases are adenosine, cytodine, thymidine, and guanosine -- some say that the initials 'ACTG' for the government AIDS clinical-trials network were not just coincidence.) In DNA the bases are paired, forming the famous double helix; cytidine always pairs with guanosine, and thymidine always pairs with adenosine.

    KP-1212 can replace cytidine when the viral enzyme reverse transcriptase is building a new copy of HIV, and pair normally with guanosine. It does not terminate the DNA chain. But KP-1212 was chemically designed to be a flexible molecule, such that it can also look like thymidine and then pair with adenosine. This introduces an error that then is locked into the viral DNA.

    These errors happen at random, anywhere in the virus; and when they do not kill the virus outright, they accumulate over generations in the DNA of the viral population. The result is eventually an error catastrophe that can wipe out the entire quasispecies, at least in laboratory tests. If you then take the drug away, the virus does not come back. And the cells on which the virus grew are still alive -- cured of the infection.

    AZT and the other approved nucleoside analogs terminate the growth of the DNA chain, killing the copy of virus being built. But that copy is easily replaced by other copies that do not have an abnormal error accumulation, so the population as a whole is not damaged. In contrast, KP-1212 continues to add new errors to the population, in addition to the errors that are already there due to the very high normal mutation rate of HIV."
  15. _Kim_

    _Kim_ Guest

    Hmmm, I've read so much tonight and it's all started to blurr, but I think I remember reading that in the latter sample, they found antibodies in 98%
  16. SDD1244

    SDD1244 Guest

  17. cfs since 1998

    cfs since 1998 *****

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    From what I have read, the 67% is from finding virus genetic material and the higher percentage is from antibody testing.
  18. acer2000

    acer2000 Senior Member

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    Right, they don't say anything about if they tested "controls" for antibodies. Thats important. Antibodies are different than finding the actual virus. What if 98% of controls have antibodies too?
  19. caledonia

    caledonia

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    Hillary Johnson's Blog / Dannybex

    She rips the CAA a new one over this, and also of course, the CDC. Worth reading:
    http://oslersweb.com/blog.htm?post=638469

    Dannybex - I had the exact same thoughts regarding the girl's basketball team, the orchestra members, etc. - lol. They don't seem to fit the modus operandi of XMRV.

    Hillary Johnson mentions an interesting thing in her blog that HIV is a slow retrovirus, which XMRV is not. So XMRV wouldn't be a long simmering thing that would pop up after 20 years, like HIV. But then again, that doesn't explain the 4% of healthy controls with XMRV.
  20. _Kim_

    _Kim_ Guest

    The best line from Hillary Johnson:

    For months, the team at WPI has been playing around with names. In lighter moments, their favorite became I.T.V.S., the acronym for, Its the Virus, Stupid.

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