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Restricted Replication of XMRV in Pigtailed Macaques

Discussion in 'XMRV Research and Replication Studies' started by Jemal, Jan 12, 2012.

  1. Jemal

    Jemal Senior Member

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    I agree it's highly unlikely they would sabotage their own experiment. They are looking for an animal model and groping around in the dark though. Nobody knows if 119 days is enough, if these monkeys can even get sick from XMRV, if some kind of trigger is needed, etc. I applaud their efforts, but let's not draw conclusions too soon...
    currer likes this.
  2. barbc56

    barbc56 Senior Member

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    I would have to disagree with this. Good scientist don't just take a shot in the dark. There has to be a priori or plausability before a study is started.These scientist work with macaques, know how viruses respond in them and how long it would take to make conclusions. I think if there is any difficulty with drawing conclusions, it's if you can compare the macaques with humans.

    Vincent Rancinello gives a good summary of the original study and has some criticisms of his own. He makes some very valid points. You can find this article here:

    http://www.virology.ws/2011/02/17/xmrv-infection-of-rhesus-macaques/

    I also want to go back and see if some of his questions were answered in this present study since the first macaque study was pre the BWG.
  3. alex3619

    alex3619 Senior Member

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    Hi barbc56, I agree and disagree. In general your statement is probably correct, but XMRV is not a well understood virus. There is therefore no model to work from, no comparison for drawing conclusions. Any conclusion is at best an educated guess. Not all viruses are equivalent. Bye, Alex
    Jemal likes this.
  4. currer

    currer Senior Member

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    "We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins"

    from Severe restriction of XMRV replication and spread... Chaipan

    The argument that XMRV or another similar virus cannot be the infectious agent causing ME BECAUSE IT IS NOT INFECTIOUS ENOUGH is laughable.

    This is precisely the type of viral effect we need to look for. ME causes lifetime disability BUT DOES NOT KILL OR LEAD TO OTHER SEVERE PATHOLOGIES with the sufferer living a normal-ish lifespan.

    We need to look for an organism that cannot be eliminated from the body but has a low virulence.

    These MLVs were thought to be harmless to humans. And they (mostly ) are!
    That is why no adequate precautions were taken against them and their pathologic potential was irresponsibly ignored for years.
    But not harmless enough.
    Long, slow, lifetime - debilitating conditions are just as unpleasant as short virulent efficient ones, as we know to our cost.
  5. Jemal

    Jemal Senior Member

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    Hi barb,

    Yes, drawing conclusions might be difficult because these are monkeys and not humans (but you have to start somewhere I guess). And like Alex said, XMRV is still a new virus and we don't fully understand it yet. So they are groping around in the dark, at least for a bit I think. Which is fine of course, the more original work a scientist does, the more difficult it becomes to set up the right experiments and draw the correct conclusions. Maybe they are making assumptions based on their knowledge of other viruses, but there's an unknown component. I just urge caution and would like to see research continue.
    currer likes this.
  6. natasa778

    natasa778 Senior Member

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    Barb I was thinking more or less the opposite, where the HGRVs would incapacitate human immune system - not in HIV-like manner, but throw it out of balance. For that I guess you would need this initial low level infection to reactivate (maybe after years and years), spread to new tissue/immune cells etc. This scenario would suit opportunistic infections AND the resident HGRV.

    I think it is downright ridiculous for authors or anyone to claim that low level infection would be harmless in humans. (---- the only way to convince me would be if 1000+ virologists went back in time, infected themselves in wombs with various HGRVs, grew up to be healthy adults and reached healthy old age with no trace of active virus, and no diseases that could be linked in any way to these viruses. I would also need to see they children grow up healthy, without horizontal or vertical spread, and neurological or immune consequences ;-)

    What I would really like to see is what happens to NK cells, mast cells and B cells after a long term ('harmless!') infection and repeated reactivation....

    I disagree that the authors would be driven by "award winning attention if they did find xmrv is harmful, so I think it very unlikely, they would sabotage. their own experiment". Virologists, esp those working with MulVs or rodent cells lines for years, would need to answer some very very tough questions if they find hgrvs harmful to humans.
    currer likes this.
  7. Firestormm

    Firestormm Senior Member

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    I refer back to the cockroach analogy Barb. The virus when injected is largely destroyed but what survives can't get out and go on to infect others.

    This study was similar to the first in that respect. It might however explain things better with regards to the virus that is not destroyed and what happens to it.

    Now whether XMRV becomes (another) EBV present in animals and indeed humans is a different story I guess. That would depend on it being present of course and until such time as another study makes a similar claim to that of Lombardi et al. the matter remains speculative at best.

    As I said though I am still reading as well, and obtaining interpretations that I can understand from those who deal with such things so can't really comment any further at this point.

    There are reasons why macaques are used in such studies when humans cannot - as you know of course - and APOBEC is I believe one of them.

    That these observations were made in non-humans is no reason for people to draw the conclusion that it is irrelevant of course.

    I hope you got my email btw.
  8. barbc56

    barbc56 Senior Member

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    Ah, thanks for reminding me about the cockroach motel analogy: they can check in but they can't check out. There's also a song called Hotel California that has this phrase. :>)
  9. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    The first macaque experiments showed that although the virus did retreat from the blood into tissue reservoirs, on reactivation through an amplification event (simulated by reinfection), the virus surprisingly rapidly ramped up. The suggestion being that there is immune system impairment that allows this to happen - the authors referred to it as some sort of immune memory (my guess is it is something to do with B-cell production). Now if this is the case, any sort of amplification event such as stress, a secondary infection, etc could have this effect. During an amplification event it is reasonable to assume that the host is contagious. So the Coffin claims are only correct until amplification occurs. At best the conclusions of the study are misleading.

    Amplification most closely fits the experiences of many patients where confusion exists about sudden rapid onset and gradual onset, with the most probable case being that both explanations coexist. It may be that only on amplification that most symptoms are realised. But in reality gradual onset occur over an extended period, may be weeks to years, and sudden onset represented by the amplification event, either through reinfection of more MRV or a secondary virus, will occur in days with vigorous intensity.

    This was probably the most significant point of the first macaque study, yet Coffin did not test it: because it would have negated most of the politically motivated statements made in the study.

    The Coffin macaque study is science, sure. But it is science by omission.
  10. alex3619

    alex3619 Senior Member

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    Hi RustyJ, I mostly agree with your analysis. I am not sure that Coffin has done anything wrong however. It is up to others to challenge these ideas: there should be two or more voices in this, not one, and I hope that further studies will be done by others. Coffin is producing data that confirms his take on things. He is not questioning and testing his hypotheses sufficiently. However this does not have to be from flaw in methodology or flaw in reasoning: a flaw in the funding system alone could explain it. Experiments have to be scoped, and rarely is there enough money to properly scope a study, it has to be more selective than that.

    Bye, Alex
  11. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    Hi Alex. I am sure Coffin could find plenty of reasons why he didn't test what must be a reasonable mechanism for the etiology of the disease, but lack of funding would not be one that I would accept; this has not been a problem in the past with the number of studies testing the contamination theory. In my opinion this was a study aimed at downplaying the Hackett study, hence undertaken with the backing of the 'establishment'.

    The question that should be asked is what does this study add or subtract to the Hackett study? In terms of new information, nothing; it tends to support the findings, point for point, so it is a validation. In terms of conclusions however it differs, downplaying the threat of XMRV on a number of fronts. It is not such a big step to assume that this was in fact the aim of the study.

    I am reminded of Dodd's comment at the 2010 Zagreb Blood Conference: In the context of XMRV, I think that there is an emergency, but it's a perceptual emergency. And I'm not as well versed in the tools of managing that, but I think that what we need to do is to manage people's reactions rather than people's safety at this point.
    currer likes this.
  12. alex3619

    alex3619 Senior Member

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    Hi RustyJ, I am not convinced that Coffin is doing what you suggest, although I would not be surprised if he was. However, I do think it will be played the way you suggest by many parties - there is a long track record of exactlly that kind of spin. Its the spin that is likely to be put on this by those who want to maintain the status quo. And for us the status quo still means disability, poverty, isolation, pain, suffering, and an early death. Bye, Alex
    currer likes this.
  13. Firestormm

    Firestormm Senior Member

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    Morning Rusty,

    I have now acquired the first paper and am having a closer look. I don't recall this but am looking. Also I wouldn't say the virus 'retreated' either but hope to add some flesh to that one later.

    Lol I still haven't finished with this Coffin paper yet! We seem to have had a batch of things XMRV-related all of a sudden and I find it hard keeping up let alone interpreting the darn things!

    Fire
  14. Firestormm

    Firestormm Senior Member

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    Hi Rusty,

    Me again I am afraid. I was just finishing my read through of that paper from American Association of Blood Banks: http://forums.phoenixrising.me/show...hreat-to-the-blood-supply&p=233680#post233680 and thought this part relating to XMRV and it's inability to function in blood might help:

    'XMRV also does not appear to be a concern for blood recipient safety. Studies have recently demonstrated that XMRV would not be able to persist or replicate in human blood due to cell-mediated antiviral pathways.9

    A large recent study further demonstrated that no XMRV antibody could be detected from 17,249 blood donors or recipients, including 13,399 US blood donors from six different regions and 3741 donors linked to 109 recipients of which 830 samples were tested over a 2-year period.

    A positive antibody result required reactivity to three different XMRV proteins, and the tests used were the same as those used by the SRWG and represented those tests that were automated and could be used for blood donation screening if needed.

    Since RNA could also not be found in any recipient or any donor with isolated antibody reactivity, the study concludes that XMRV is not a current threat to blood safety.15'

    9. Knox K, Carrigan D, Simmons G, Teque F, Zhou Y, Hackett J Jr, Qiu X, Luk KC, Schochetman G, Knox A, Kogelnik AM, Levy JA. No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected. Science 2011;333:94-7.

    15. Dodd RY, Hackett J, Linnen JM, Dorsey K, Wu Y, Zou S, Qiu X, Swanson P, Schochetman G, Gao K, Carrick JM, Krysztof
    DE, Stramer SL. Xenotropic murine leukemia virus-related virus (XMRV) does not pose a risk to blood recipient safety. Transfusion 2012;52:298-306.

    As I said on the other thread - I totally forgot about the paper from Dodd! Hard to remember much these days unless it keeps getting reinforced...
  15. currer

    currer Senior Member

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    We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication.

    However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins"

    from Severe restriction of XMRV replication and spread... Chaipan
    http://www.ncbi.nlm.nih.gov/pubmed/21325415
  16. Firestormm

    Firestormm Senior Member

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    Thanks Currer - do you mean this paper: Inhibition of xenotropic murine leukemia virus-related virus by APOBEC3 proteins and antiviral drugs http://www.ncbi.nlm.nih.gov/pubmed/20335265? If not can you post a link and I will take a look.
  17. currer

    currer Senior Member

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    http://www.ncbi.nlm.nih.gov/pubmed/21325415

    "We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins.........

    .....However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors"
  18. alex3619

    alex3619 Senior Member

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    Yes currer, restriction factors slow and reduce infection, they don't stop it. So it will result in decreased infection rates, and a slower spread of infection in a host, and perhaps mostly restrict the infection to more optimum tissue types. It does not stop infection. Only lack of receptors that allow entry can do that. This is in accordance with other viral findings. A claim of non-infectivity due to restriction factors is not plausible. One thing it might do though is reduce infection via blood plasma (cell free blood products) to almost zero. Bye, Alex
  19. Firestormm

    Firestormm Senior Member

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    I know some folk don't read ERV but this one about the Rhesus studies mentioned in this thread (apart from the Coffin/Pathak study which is the topic) were covered very well here last year: http://scienceblogs.com/erv/2011/02/xmrv_and_human_pbmcs_do_not_wa.php

    In relation to the Chaipan paper: http://www.ncbi.nlm.nih.gov/pubmed/21325415 but also in relation to the other paper from Onlamoon http://www.ncbi.nlm.nih.gov/pubmed/21325416, Abbie was saying the following that I think pertinent as it applies just as much to the latest paper from Coffin which largely said the same as Onlamoon (though I have yet to read it all through myself yet [sigh]):

    XMRV and human PBMCs: DO NOT WANT!!!!!!!! February 2011

    'Long story super-short: Human PBMCs to XMRV: DO NOT WANT!!!!!!

    Long story short: Human PBMC (white blood cells) dont want or like XMRV. They are very inefficiently infected, and when they are infected, they produce very little virus. The little virus they do produce is hypermutated, and almost certainly abortive.

    Long story not short at all:

    The focus of this paper [Chaipan Feb 2011] is a group of proteins every cell in your body has to defend themselves against retroviruses (want me to make an evolutionary guess, they protect us from wayward ERVs)-- APOBEC (Wikipedia page for a specific one, A3G).

    APOBEC is packaged in babby viruses, and then when the babby goes on to infect a new cell, APOBEC screws up their reverse transcription process and makes a ton of mutations in the new proviral DNA. You might say "OY! ERV! Retroviruses mutate all the time anyway, rite? So why do they give a crap if ABOBEC makes them mutate more? Wouldnt that be better for a retrovirus?"

    While Creationists (and other anti-/non-science groups) might present viruses as these mindless mutating machines, the number of 'mistakes' that happen during reverse transcription is a evolved number. It makes those mistakes because they provide the ability to explore as much sequence space as possible... without making so many mistakes that it just wont work as a virus anymore (error threshold).

    APOBEC pushes retroviruses over that error threshold (unless the virus has evolved a protein to counter the APOBEC).

    So what happens is-- A retrovirus productively infects a cell. The infected cell produces lots of babby viruses. APOBEC stows away in those babby viruses, which go on to infect new cells... BUT! During reverse transcription, APOBEC introduces lots of unwelcome mutations. Which means even if that new cell is 'infected', the virus that inserts into its genome is crap. It cant make any more babby viruses.

    APOBEC is like the retroviral version of Cockroach Hotel: Retroviruses can check in, but they dont check out.


    An obvious observation is that XMRV has no apparent counter to human APOBEC, but it might. But previous experiments didnt seem to indicate there was one. ?

    So... if XMRV is infecting humans... how is it getting around our APOBECs? If XMRV is not getting around our APOBECs... is XMRV infecting humans?

    These folks started in cell lines. As Fraulein Maria would say 'A very good place, to start!' They infected 10 billion CEM cells (a T-cell line that expresses a lot of APOBEC3G and 3F) and 10 billion CEM-SS cells (a related T-cell line that expresses little/no A3G or A3F) with either 10^4, 10^5, 10^6, 10^7, 10^8, or 10^10 XMRV viruses (based off RNA copies/volume).

    At 10^4, not a damn cell was infected, either line. But when they used more virus, the CEM-SS cells were infected and made lots and lots of baby viruses, while the CEM cells exposed to the same quantity of virus, were like 'Meh.' They got infected, they made *some* babby viruses... but those babby viruses didnt really go on to infect more cells to produce MORE babby viruses.

    'This result is consistent with the expectation that most of the proviruses in the CEM cells will be hypermutated and will be unable to produce viral particles.'

    CEMs, CEMs, a human T-cell line, Cockroach XMRV hotel.

    But cell lines are not 'human'. So the next step was to isolate white blood cells from a few different people (one of the putative targets of XMRV, which express lots of A3G and A3F), made 10 billion cell aliquots of those cells, and did experiments with them.

    The cells pretty much wanted nothing to do with XMRV. There's not much else to say. They did the same thing as the CEMs-- some of the cells were infected, but they didnt *produce* babby viruses:

    'Importantly, we did not observe any increase in viral RNA copies in the infected PBMCs during the next 15 days, indicating that there was little or no virus replication and spread. '

    If the cells are good targets, you expect to put on 'some' virus, and get out A TON OF VIRUS. Didn't happen here.


    Furthermore, the few babby viruses that were made were mutated by APOBEC3G and 3F. They sequenced the babby viruses and saw A3G/A3Fs mutational signatures. Actually, they only sequenced a ~1200 nt region of the virus, and they saw more mutations in viruses we *know* came from a clonal stock than I see in the 'XMRV sequences' uploaded to Genbank, which theoretically come from 'wild type totally in nature' variants.

    lulz.

    They also made a neat indicator cell-based 'test'. Mix 100,000 PBMC from someone with their indicator cells. If at least 7 of those cells are infected with XMRV or any other MLV-like-creature, their test will see it. No PCR with magic primers. No flow that could be non-specific. No antibodies that could be non-specific. Just: Is there a gammavirus up in here or not? Are the indicator cells green or not?

    While that is neat, there is one more thing I would have wanted in this paper-- XMRV RNA is not increasing in infected PBMC cultures (not getting multiple rounds of infection). I would have liked to have seen PBMC supernatant filtered, and put on, say, the CEM-SS. I dont care if the CEM-SS are infected. They should be. I want to know if XMRV RNA increased in *THAT* culture. How much of the crap virus coming off the PBMC is still functional? I dont think they have the data to say this:

    'Although XMRV replication and spread in PHA-activated PBMCs was severely restricted, PBMCs could serve as a reservoir of replication-competent XMRV and facilitate infection of A3G/A3F-deficient cells.'

    Yes, they 'could', but it would have been nice to have some data to see how 'couldliest' that possibility is. But maybe they did and I just dont totally 'get' their indicator cells.


    Thats the worst thing I can say about this paper. Its straight forward. The data moves the field forward. HUZZAH!

    So why the hell was I ranting about animal research in the beginning?

    Because the data in this paper could have helped this paper [Onlamoon February 2011]. It could have made the deaths of those eight rhesus macaques most likely to yield the largest quantity of data/life.

    As is, the take-away message from that paper is "If you pump 10^6 XMRV IV into macaques (a dose that would leave you with a pile of dead monkey with SHIVs), pretty much nothing happens.

    No clinical symptoms. The XMRV gets in, but there isn't much (any?) productive infection." Where did the >10^6 number come from? What was the logic? Why not 10? Why not 10^10? 10^6 is a dead-monkey-dose with SHIV, but 10^6 XMRV on human isolated PBMC did jack shit. So why would you put 10^6 in an animal?


    Think of that this way-- lets say you put a few cells of algae in a fish tank, and they didn't take hold to make your fish tank gross. Why would you take that same 'dose' of algae and expect it to take hold in the Colorado River? And when the hell is any human exposed to >10^6 XMRV IV in real life? Wat?

    And Im looking at previous XMRV publications that indicated APOBEC is a Big Deal with XMRV restriction. At least with HIV-1, human APOBEC, human A3G is, what, 100-fold more potent than macaque A3G? Where's the macaque in vitro data, like the data that is the topic of this post? They talk about APOBEC all over the place, but didn't feel it was necessary to investigate human vs macaque in vitro before they decided they made an animal model?? Wtf?

    That paper is a rush job that gave us very little information in exchange for 8 dead monkeys, before basic bench research was done.

    We can do better.'
  20. currer

    currer Senior Member

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    Tabloid virology, firestormm.

    I like to think we are broadsheet here!

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