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Susceptibility of (XMRV) to retroviral restriction factors

Discussion in 'XMRV Research and Replication Studies' started by RedRuth, Jul 23, 2011.

  1. RedRuth

    RedRuth Senior Member

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    Sorry I can't find it, maybe I'm looking in the wrong place however the second JVI paper only came out in May 2011 so presumably she hasn't addressed their results.

    Almost certainly that's the way these viruses have spread. Interestingly I found out while looking at another site that a cell we have is infected with an MLV. It's bad practise to have different cell types in the hood at the same time but I expect most people have done it at some time.
  2. RedRuth

    RedRuth Senior Member

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    I really doubt if this can be spread as an airbourne disease. It's not the same thing as contaminating cell cultures with aerosols.
  3. mellster

    mellster Marco

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    First of all I appreciate sometimes or even often dissenting voices like RR on this board - we need to challenge our own convictions daily. I'd really like to know from Ruth what you think is the most likely origin(s)/cause(s) of ME/CFSID at this point, taking current science and its findings so far into account?
  4. currer

    currer Senior Member

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    Hi, Redruth,

    The thread is at http://forums.phoenixrising.me/show...istics, or whether is has unique properties..
  5. RedRuth

    RedRuth Senior Member

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    Well I'm not an epidemiologist or a virologist so my opinion is not worth much. But, it seems extremely likely that there is at least a viral component to ME, but XMRV is a retrovirus and just doesn't fit, there's no suggestion ME is sexually transmitted or any of the ways other retroviruses are spread. I would have thought one of the herpes viruses would be a more likely candidate. Something that most people are exposed to but only affects some people so adversely, either because of genetics or environmental factors.

    Some of the other problems with XMRV (if it isn't contamination) being a cause are the host cell restriction factors that we're discussing here and the finding that the complement system was able to inactivate XMRV. It may turn out that XMRV is present in ME suffers but this doesn't mean it causes ME, far from it (correlation does not imply causation).
  6. ukxmrv

    ukxmrv Senior Member

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  7. ukxmrv

    ukxmrv Senior Member

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    B cell lines have a mature CD20+, CD23+ phenotype and produce infectious XMRV. Virus production occurred despite extensive hypermutation of the proviruses in these cells by APOBEC3G. Therefore, XMRV infection may accelerate the development of B cell malignancies by either indirect chronic stimulation of the immune system and/or by direct infection of the B-cell lineage. Since viral load in peripheral blood is low, these data suggest that B cells in tissues such as spleen and lymph nodes could be an in vivo reservoir for XMRV.


    from http://forums.phoenixrising.me/showthread.php?10440-XMRV-amp-Blood-Supply-Webinar-Tuesday-Mar-29 (and thanks to Lansbergen)

    and

    6. Something Lipkin had also noticed, copathogens can increase severity.

    7. Patient contacts including caretakers have higher risk of XMRV so this might be considered as a screening criteria for blood donations.

    8. A question about XMRV transmission noted that an implication of CROI is that XMRV might be airborne. This is being investigated, but is not proven.

    9. Mikovits is talking to Dr. Lerner about XMRV and herpes virus interactions.

    10. Mikovits describes ME/CFS as an Aquired Immune Deficiency.

    11. A new high security lab may enable research they have not been able to do so far.

    12. It was confirmed that Chronix have indeed claimed to have found fully integrated XMRV in ME/CFS, including flanking DNA on both sides of the virus.

    13. Pathogenicity is not proven. However, it is suspected that antivirual methylation might be removed by epigenetic factors including those induced by the virus itself.


    (thanks to Alex, http://forums.phoenixrising.me/show...amp-Blood-Supply-Webinar-Tuesday-Mar-29/page5 )

    and

    https://picasaweb.google.com/magnet...AMikovits?feat=directlink#5589595830298241410


    (slide from Dr Mikovits about ABOBEC3G)
  8. RedRuth

    RedRuth Senior Member

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    Thanks, I'll take a look.

    Really? I find this difficult to believe. The XMRV genome is smaller than the HIV genome why would this make it more stable? The reason retroviruses are unstable outside of body fluids is that
    1. They only have an envelope made of host cell membrane plus the env protein (and possibly some host cell proteins) which won't keep it's integrity in the air or water for very long or anywhere out of it's usual environment.
    2. They're RNA viruses, our skin secretes RNAse enzymes that very quickly degrade RNA. Ask nayone who has tried to extract RNA about RNAse enzymes and their stability and efficiency.

    Regarding aerosols, they only have to survive long enough to get into another tissue culture flask with media in it, in other words, not very long and they don't have to get past the innate immune system. If I get an aerosol on my hands (unlikely given that I always wear gloves) any cells or retrovirus particles won't last long (see above).

    Yes, definitely.
  9. RedRuth

    RedRuth Senior Member

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    Not really, APOBEC is active in PBMCs and tetherin is active in all hematopoietic blood cells. This is why the Groom et al paper and the JVI paper are at odds with the Lombardi paper.

    EDIT: I think that maybe whoever wrote that comment is confusing hypermutation (which is the strategy APOBEC uses for inactivating viruses) with the phylogenetic analysis of the viral sequences.
  10. RedRuth

    RedRuth Senior Member

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    Thanks but that's from a meeting abstract, has it been published? I'll give it a better read when I've more time but it doesn't look too different from the JVI paper.
  11. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    I think its important to remember Mikovits has repeatedly referred to B cells as an XMRV reservoir, for quite some time, although I don't have links to any research she may have been referring to. There is this:

    http://www.retroconference.org/2 011/Abstracts/40987.htm


    Session 43-Themed Discussion
    TD: XMRV: New Findings and Controversies
    Wednesday, 1-2 pm; Room 302-304
    Paper # 217
    Presence of XMRV Sequences in B Cells Are Restricted by APOBEC
    Jorge Carrillo1, J Blanco1, E Garcia1, J Arreal2, B Clotet1, and C Cabrera1
    1Fndn irsiCaixa, Barcelona, Spain and 2Hosp Univ Germans Trias i Pujol, Barcelona, Spain

    Background: A link between xenotropic murine leukemia virus-related virus (XMRV) infection and different human diseases, such as chronic fatigue syndrome (CFS) and prostate cancer, has been recently established. Given that this retrovirus can infect different tissues and cell types both in vivo and in vitro, including cells from the immune system, the identification of the cellular compartment(s) where XMRV can establish a reservoir may be useful to understand the pathology associated with this virus.

    Methods: In order to determine the presence of XMRV in B lymphocytes, we have screened EBV-transformed B-cell lines available in our laboratory. The origin of these cell lines include CFS patients (n = 11; fulfilling both Fukuda and Canadian criteria), HIV+ individuals (n = 4), prostate cancer patients (n = 1), and healthy donors (n = 5). DNA was extracted from cellular dry pellets and several XMRV genes were amplified by either real-time PCR (pol) or nested PCR (gag and env).

    Results: We detected 7 positive samples from 14 individuals tested by RT-PCR (4 CFS, 2 donors, and 1 HIV+ individual). env amplification identified 4 positive samples out of 21 individuals tested (3 CFS-affected individuals and 1 healthy donor), whereas gag amplification showed only 3 positive samples (1 CFS-affected individual, 1 healthy donor, and one HIV+ patient). To confirm the presence of XMRV we performed sequence analyses of gag and env amplicons. The 3 gag sequences available showed a 100% homology with XMRV gag published sequences, including the XMRV characteristic 24nt deletion which is not found in any known exogenous murine leukemia virus. Furthermore, env sequences were also homologous to previously described XMRV sequences, showing none or low variability. Interestingly, most of the observed changes corresponded to multiple G to A mutations that were accumulated in 1 positive sample, resulting in a truncated env protein and suggesting that APOBEC-related restrictions operate in vivo during XMRV infection.

    Conclusions: Despite the discrepancies observed in the different PCR approaches, our data provide evidence that EBV-transformed B-cell lines harbor XMRV-specific sequences, although the establishment of this infection could be modulated by the innate restriction factor APOBEC 3G . Our data suggest that, in vivo, B cells may represent a reservoir for XMRV contributing to its potential pathogenesis.
  12. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    The same post ukxmrv referred to had this comment by Judy.

    There are many MLV studies on mice showing pathogenicity: neurological disease, cancers etc. Of course mice don't have the same restrictions, but you should start having suspicions.
  13. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    Human APOBEC3 proteins can inhibit xenotropic murine leukemia virus-related virus infectivity

    Hal P. Bogerda, Fengwen Zhangb, Paul D. Bieniaszb and Bryan R. Cullena, low asterisk, E-mail The Corresponding Author

    a Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, NC 27710, USA

    b Aaron Diamond AIDS Research Center, Laboratory of Retrovirology and Howard Hughes Medical Institute, the Rockefeller University, New York, NY 10016, USA
    Received 13 September 2010;
    revised 25 October 2010;
    accepted 10 November 2010.
    Available online 4 December 2010.

    Abstract

    Xenotropic murine leukemia virus-related virus (XMRV) is a novel retrovirus, related to murine leukemia virus (MLV), that has been implicated in human disease. If XMRV is indeed able to replicate in humans, then one might predict that XMRV would have developed resistance to human innate antiviral resistance factors such as APOBEC3G (hA3G). In fact, we observed that XMRV and MLV are both highly sensitive to inhibition by hA3G and equally resistant to inhibition by murine APOBEC3. While several human prostate cancer cell lines were found to lack hA3G, stable expression of physiological levels of hA3G rendered these cells refractory to XMRV replication. Few human tissues fail to express hA3G, and we therefore hypothesize that XMRV replicates in one or more hA3G-negative reservoir tissues and/or that human XMRV infections are likely to be rare and potentially of zoonotic origin.

    http://www.sciencedirect.com/science/article/pii/S0042682210007270
  14. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    J Virol. 2010 Oct;84(20):10933-6. Epub 2010 Aug 11.
    The glycosylated Gag protein of a murine leukemia virus inhibits the antiretroviral function of APOBEC3.
    Kolokithas A, Rosenke K, Malik F, Hendrick D, Swanson L, Santiago ML, Portis JL, Hasenkrug KJ, Evans LH.
    Source

    Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA.
    Abstract

    APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.

    http://www.ncbi.nlm.nih.gov/pubmed/20702647
  15. RedRuth

    RedRuth Senior Member

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    You probably should have highlighted the whole of that sentence we therefore hypothesize that XMRV replicates in one or more hA3G-negative reservoir tissues* and/or that human XMRV infections are likely to be rare and potentially of zoonotic origin. This is another paper that underlines how unlikely replication of XMRV is in Human cells. It also would seem to imply that MLV's can't evade Human APOBEC

    * The recent JVI paper found that hA3G negative cells efficiently restricted XMRV presumably because they express tetherin.
  16. currer

    currer Senior Member

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    Hi, redruth,

    There are some instances of ME being passed on sexually, but as no adequate research has been ever done on this disease, we are reduced to anecdotal cases.

    However there is much more evidence for parent to child transmission, if that is what is happening.
    A worrying development that I noticed, and that my GP also has noticed, is that ME has become more common in children. When ME first became common it was noted in adults, but now appears in much younger age groups, often where the mother has ME.

    So the pattern is - first generation - adults,
    second generation offspring of affected adults.

    This has been reported ion the US and I have a friend where this exact pattern has occurred.

    Why do you think this might be?

    XMRV was first discovered because researchers were looking for a sexually transmitted agent in prostate cancer. (analogous to HPV and cervical cancer) This is because the rates of prostate cancer over the past thirty years have tripled in all age groups, (not only the elderly) So they were looking for a sexually transmissible agent. XMRV has been found in semen. (sorry cant look for the reference, perhaps someone else can ferret it out)

    Because retroviruses cannot be eliminated from the body, I would think they would be a likely agent to spark the development of an auto immune type disease. Infected cells would express XMRV proteins and the immune system would recognise them as such. XMRV would not need to be directly pathogenic for it to have a deleterious effect on its host.
    (The relevant paper here is probably the macaque study) This showed that although XMRV cleared rapidly from the blood, an immune challenge reactivated virus from tissue into the bloodstream.)

    I dont think anyone would want a new retrovirus in them, however inert it was thought to be.
  17. currer

    currer Senior Member

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    [QUOTE=RedRuth;195246



    Really? I find this difficult to believe. The XMRV genome is smaller than the HIV genome why would this make it more stable? The reason retroviruses are unstable outside of body fluids is that
    1. They only have an envelope made of host cell membrane plus the env protein (and possibly some host cell proteins) which won't keep it's integrity in the air or water for very long or anywhere out of it's usual environment.
    2. They're RNA viruses, our skin secretes RNAse enzymes that very quickly degrade RNA. Ask nayone who has tried to extract RNA about RNAse enzymes and their stability and efficiency.

    Regarding aerosols, they only have to survive long enough to get into another tissue culture flask with media in it, in other words, not very long and they don't have to get past the innate immune system. If I get an aerosol on my hands (unlikely given that I always wear gloves) any cells or retrovirus particles won't last long (see above).



    Dealing with your last points first, it sounds as if you are arguing against the likelihood of contagious spread of this virus as it cannot get past the body's defences.
    I suppose this would make the likelihood of spread of XMRV via artificial or blood borne means more likely, if XMRV is found in humans. I do not prefer to argue for one or the other means of transfer. I am only exploring possibilities and using my memory of discussions I have heard among virologists over the past year. I remember now that JM said simple, not small (my mistake).


    With regard to the argument about restriction, my feeling is that all the theoretical ideas about why xmrv cannot replicate in people fail, if xmrv is in fact found in people. In this case it is the theory that has to change and not the virus. So no amount of theoretical argument can override a factual finding. We then are forced to adjust our theoretical understanding in the face of reality.
    From what I have read about restriction factors recently, it seems there is much that is not yet understood on how viruses adapt to the cells they use.

    (assuming xmrv really is there, of course!)
  18. RedRuth

    RedRuth Senior Member

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    All good points and I haven't read the Macaque study yet. I suppose if the children of ME suffers also get ME then it may imply an infectious agent or a genetic susceptibility. I really don't know, what do epidemiologists say about the spread of the disease?
  19. RedRuth

    RedRuth Senior Member

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    Yes, that's right.

    EDIT: To clarify I think (with all the caveats about the worth of my opinion) that you have a plausible hypothesis. XMRV is a real virus (not disputed) but if it is in the Human population then it can't replicate efficiently like HIV so it may follow an infective pathway more like HTLV-1.

    EDIT for your edit!: Lol our edits are crossing: I'm not necessarily saying that XMRV can't infect Humans because of the restriction factors it's just that the cells that the original Lomabardi paper found XMRV protein in, PBMCs, express both APOBEC and tetherin and efficiently restrict XMRV, therefore there won't be detectable XMRV proteins. This was the point the recent JVI paper was making.

  20. Marco

    Marco Old blackguard

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    How would they know if they can't even agree on a standard robust definition?

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