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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. barbc56

    barbc56 Senior Member

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    Were the macaques in the present study destroyed? I have been going through so many papers my head is a spinning. :>)
     
  2. currer

    currer Senior Member

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    Well they looked at their tissues....prostate and LN (lymph nodes?)
     
  3. barbc56

    barbc56 Senior Member

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    Facepalm. Of course.

    I just found it in the full length paper. Poor things.
     
  4. barbc56

    barbc56 Senior Member

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    Currer wrote"

    Forum meme where assumptions spread without scientific backing, could also be considered tabloid virology.

    Whether you like Abbey's style or not, she is a credible scientiest.
     
  5. Jemal

    Jemal Senior Member

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    As I said, sucks to be macaques, at least if you are stuck in some lab!
     
  6. natasa778

    natasa778 Senior Member

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    ERV is either clueless/missing some very important information, or spinning truth on purpose. Not sure which of the two would make her worth listening to?

    She is failing to mention one little detail:

    http://www.ncbi.nlm.nih.gov/pubmed/22145963

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671846/?tool=pubmed

    http://www.ncbi.nlm.nih.gov/pubmed/15286366


    So much for her being a "credible" scientist....
     
  7. natasa778

    natasa778 Senior Member

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    oh, and there is one other thingy that "credible" "scientist" blogger "fails" to mention in her rants.

     
  8. barbc56

    barbc56 Senior Member

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    Natasha, why do you think these statements are not credible. Thanks.
     
  9. natasa778

    natasa778 Senior Member

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    Because ERV's statements are opinions/conclusions based on seeing only tiny (self-selected) part of the picture, through her ignorance or spin.
     
  10. Firestormm

    Firestormm Guest

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    I am wondering if in fact this paper says the same as what Abbie was herself concluding - that the virus which 'survives' is so mutated as to render it neutered:

    From the paper abstract: 'Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV.'

    I will continue to read it through though...
     
  11. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    The statement is a suggestion only, more like a theory, and does not exclude an assumption that XMRV can get by the block. Mutated virus has not been proven to be harmless. There is some research that 'suggests' virus particles can cause the inflammation issues. In the Hackett study virus particles were found in the brain, some time after infection. This implies at the very least that virus is still being produced somewhere, even if it is then being hypermutated. Either that or the so-called neutered virus is replicated, lol?

    Was all the virus hypermutated? Some virus hypermutation is somewhat irrelevant if whole virus is still present. They found hypermutated virus, which proved A3G did operate, but A3G definitely does not happen in all tissue cells, nor is it a factor in cell mitosis, which is one way for infected cells to spread. So in a way it is impossible for A3G to do the job completely.

    The abstract is worded very carefully. Note it says 'one of the blocks'. They don't know of any others really. And they know A3G doesn't do the job 100%, so to fit their theory that the virus isn't in humans they have to say there must be other blocks. You will find this evasive sort of language all through the negative studies. Sort of like trying to squish the data to make it say what they need it to say.
     
  12. Firestormm

    Firestormm Guest

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    Thing is Rusty some folk seem to want absolutes when they deem it necessary and at other times are not as concerned. I am reading now the Coffin Pathak paper this thread is all about and will post some extracts later - but what I do post - if some don't agree for whatever reason - can be said to be 'inconclusive' or 'does not go far enough'.

    It is up to the next scientist (group of scientists) to pursue the questions. Each paper cannot be held to be absolute. As we saw with Lombardi et al for example. However, when a body of evidence is built up that all points towards there being no XMRV found in humans - then they turn to animal studies. Because XMRV is still a novel retrovirus worth looking at.

    One day XMRV might be found in humans. Might. And so they need to keep studying the retrovirus in vitro in human blood as well as in monkeys and mice etc. I see that elsewhere on the forum and on Facebook a certain group now believes Lipkin should be looking in lymphoid tissue based on one of the macaque studies.

    This is plain nuts for a couple of reasons but largely because we should have some faith in these scientists knowing what the hell they are doing, no? Also Lipkin has spent the money, and the study is about Lombardi and Lo i.e. blood in humans and not darn monkeys.

    Returning to the hypermutated virus and 'one of the blocks' there is more about this in the Coffin/Pathak paper but again - and I have no doubt - someone will say 'Yeah but they looked at ringtailed macaques and not macaques nor humans for that matter - so how can they compare like with like'!

    I mean we can look now at papers that are researching XMRV for what it is - a novel retrovirus that has not been found in humans. We can look at the papers as they come out and discuss those papers and try to interpret them in lay-language or we can 'cherry-pick' to suit our own beliefs. Or a mixture and more I guess.

    I would just like to be able to ask questions about the science-jargon and minutiae of the papers - the bits I don't understand - and seek interpretations from those who do. Individual papers can't answer all the questions but they can and do address some that have been raised before. And collectively suggest emerging patterns.

    As a further aside: What happened to those who claimed XMRV = VP62 and not worth considering? Clearly XMRV is worth considering, it is infectious and it is a retrovirus even though it originated from 22Rv1. It was just never found in humans is all.

    Anyway, and apologies, back to the Thread's original paper which is interesting (unlike my nonsense) and does indeed differ from Onlamoon's macaque study in 2011.
     
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  13. Firestormm

    Firestormm Guest

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    Some extracts I found interesting from the main paper under discussion (no minutiae as such):

    Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques

    Gregory Q. Del Prete1, Mary F. Kearney2, Jon Spindler2, Ann Wiegand2, Elena Chertova1, James D. Roser1, Jacob D. Estes1, Xing Pei Hao1, Charles M. Trubey1, Abigail Lara1, KyeongEun Lee2, Chawaree Chaipan2, Julian W. Bess, Jr.1, Kunio Nagashima3, Brandon F. Keele1, Rhonda Pung4, Jeremy Smedley4, Vinay K. Pathak2, Vineet N. KewalRamani2, John M. Coffin2,6, and Jeffrey D. Lifson1*

    Abstract

    1. ...These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread...

    Introduction

    2. In 2006, Urisman and coworkers identified sequences from a novel gammaretrovirus in an analysis of human prostate tumor tissues using a Virochip DNA microarray and named the new virus xenotropic murine leukemia virus-related virus (XMRV) due to its high sequence identity with xenotropic murine leukemia viruses (X-MLV) (64). Although several follow up studies described findings interpreted as evidence of XMRV infection in up to 27% of prostate cancer patients and up to 4% of healthy prostate controls using PCR (3, 9, 11, 54), immunohistochemistry (54), and fluorescence in situ hybridization (3) on prostate tissues, as well as anti-XMRV serum neutralization assays (3), the majority of studies detected little or no evidence for XMRV infection in either prostatic tumors or healthy controls, raising questions about the authenticity of human XMRV infection and what role, if any, XMRV might play in prostate cancer (1, 2, 15, 23, 50, 56, 61)

    3. Although there is a growing consensus that evidence for XMRV infection in human samples is more likely the result of contamination than genuine in vivo infection, the fact remains that XMRV is a novel replication-competent retrovirus of unknown pathogenic potential once implicated in the etiology of several human diseases...'

    4. '...Given uncertainties about the capacity of XMRV to cause human disease as well as the potential target cell tropism, tissue distribution, in vivo replication capacity and sequence evolution of the virus, and elicited antiviral immune responses, we infected two pigtailed macaques (Macaca nemestrina) with a well-characterized 22Rv1-produced XMRV stock. We conducted these studies with two primary goals.

    First, we sought to generate bona fide in vivo derived positive control samples for PCR, RT-PCR, and serology assays performed on clinical samples (10, 28).

    Second, we aimed to comprehensively examine the natural history of in vivo XMRV infection in a primate host, evaluating levels and kinetics of replication, viral sequence changes, cell and tissue tropism, and cellular and humoral antiviral immune responses.'

    5. 'We show here that XMRV replication in pigtailed macaques is restricted, with limited, transient viremia associated with the accumulation of extensive G-to-A hypermutation in cell-associated viral DNA. In spite of limited viral replication, humoral immune responses to the virus were relatively robust and stable, while innate immune responses were transient and adaptive cellular immune responses were negligible.

    Discussion

    6. 'Although based on accumulating results, the proposed association of XMRV infection with human disease as well as evidence of any authentic human XMRV infection appear increasingly unlikely, the virus itself is a replication-competent retrovirus of unknown pathogenic potential that is capable of infecting human cells (7, 11, 47).'

    7. 'Given the lack of any confirmed positive cases of human XMRV infection (29, 58), the in vivo replication capacity, sequence evolution, tissue tropism, and elicited immune responses associated with XMRV infection are unknown and it is unclear what results using a variety of direct and indirect detection methods might be expected in the setting of authentic XMRV infection.'

    8. 'Information about the natural history of XMRV infection and host immune responses to the virus would provide a framework to help interpret results suggesting evidence of human XMRV infection. In the absence of any known human infection, animal models must be relied upon to provide this information.'

    9. 'To address these questions in a non-human primate species, we intravenously infected two adult male pigtailed macaques with >1010 XMRV virions, an inoculum which likely exceeds by many orders of magnitude any viral inoculum that might be involved in a physiological human transmission.'

    10. 'Despite this large viral inoculum, XMRV replicated only transiently to relatively low peak levels in both animals, achieving peak plasma viral loads ? 2,200 RNA copies/ml that declined to undetectable levels within 4 weeks of infection.'

    11. 'This decline in viremia was associated with striking levels of G-to-A hypermutation in PBMC-associated vDNA, likely reflective of APOBEC-mediated viral restriction.'

    12. 'Although plasma viremia was brief, within the first 2-4 weeks of infection both animals raised robust anti-XMRV antibody responses primarily directed towards p15ETM, p30CA, and gp70SU that were largely maintained up 550 to 119 days post-infection.'

    13. 'In addition to these binding antibody responses, neutralizing antibodies were also elicited within the first two weeks of infection and maintained through the studys conclusion.'

    14. 'In contrast, innate immune responses in lymph nodes were only transiently upregulated in the first week of infection and rapidly diminished to baseline levels by two weeks post-infection, while adaptive T cell responses were essentially negligible in ICS format assays using whole XMRV virions or purified recombinant XMRV proteins as stimuli.'

    Continued (sorry)
     
  14. Firestormm

    Firestormm Guest

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    Some key observational differences between this paper and the previous macaque study from Onlamoon (Feb 2011 link is back on the thread somewhere):

    'Our findings differ markedly from some of those made in a previous study by Onlamoon and coworkers, which examined XMRV infection of rhesus macaques (Macaca mulatta).
    Although Onlamoon et al. also reported low, transient levels of viremia that declined to undetectable levels within 3 weeks of infection (albeit with a less sensitive qRT-PCR assay) associated with the induction of a relatively stable antibody response with neutralizing activity, there were several key differences between results for the rhesus macaque infection study and those reported here for pigtailed macaques.

    15. 'First, while we observed a relatively stable pool of PBMC-associated XMRV DNA composed predominantly of G-to-A hypermutated viral genomes that were detectable through 119 dpi, Onlamoon et al. reported a complete loss of PBMC-associated vDNA within the first month of infection (44). Although a recent follow-up study has shown G-to-A hypermutation in PBMC-associated vDNA sequences in these infected rhesus monkeys (67), the disappearance of vDNA in PBMCs seems to suggest that factors other than APOBEC-mediated restriction may contribute importantly to control of XMRV infection in rhesus macaques.'
    It is not clear, however, if the PCR assay used here and that used by Onlamoon and coworkers are equally capable of detecting hypermutated vDNA, which are likely less efficiently amplified due to inefficient priming, and it is possible that the apparent loss of vDNA-positive PBMCs in rhesus macaques simply reflects the accumulation of hypermutated genomes.'

    16. 'Other key differences between this and the Onlamoon et al. study were noted when comparing analyses of various host tissues. In our pigtailed macaques, we identified vDNA in LNMCs by X-SCA at early and late post-infection time points, however longitudinal LN sections were vRNA negative when probed with our ISH riboprobe cocktail, the specificity of which was verified using in vitro infected pigtailed macaque cells (see Figs. 2B and S1).
    These results were consistent with our observations in PBMCs vis--vis plasma viremia and suggest that the vDNA positive cells detected by X-SCA in LNs were not productively infected.
    Conversely, the Onlamoon et al. study reported that LNs from infected rhesus macaques contained productively infected cells detectable by immunohistochemistry (IHC) at both early and late (>140 dpi) post infection time points (44).'

    17. 'Since XMRV was initially linked to human prostate cancers, we also evaluated viral replication in prostate tissue. By X-SCA, we detected very low levels (<15 copies/million cells) of vDNA in snap frozen prostate tissue pieces collected at necropsy, and no vRNA positive cells were detectable in prostate tissue sections by ISH.'
    Since a small number of contaminating blood cells could be the source of this low level vDNA, these data indicate very limited or no XMRV infection in prostate with no productive infection at 119 dpi. Although Onlamoon and coworkers noted fewer productively infected cells in prostate tissue at late post infection time points than at early post-infection time points, they reported that vRNA+ cells could still be detected by ISH in prostate tissue >140 dpi (44).'

    Concluding remarks

    18. 'These results in rhesus macaques [from Onlamoon] suggest the establishment of a chronic viral infection, characterized by the stable presence of productively infected cells in LNs and other tissues, concomitant with the apparent loss of vDNA+ PBMCs and undetectable plasma viremia, suggesting that once the virus makes it into LNs and other tissues it continues to replicate but becomes trapped and does not reseed the peripheral blood compartment despite the apparent presence of suitable target cells.

    19. 'This conclusion is in stark contrast to the results we report here for pigtailed macaques, wherein early rounds of viral replication lead to the seeding of PBMCs and LNMCs with a stable pool of archived, hypermutated, likely non-functional viral genomes. In addition to the difference in macaque species, differences in the sensitivity and specificity of the assays employed could also contribute to the discordant results.'

    20. 'In the absence of any confirmed human XMRV infection cases, it is unclear whether infection of pigtailed macaques accurately models human XMRV infection. There is, however, evidence to suggest that several features of pigtailed macaque infection may mirror what would occur in an XMRV infected human.'

    Previous work has shown that XMRV infection of human PBMCs in vitro results in a non-spreading, restricted infection with viral production showing a dose-dependent plateau effect with extensive G-to-A hypermutation in cell associated vDNA, likely mediated by APOBEC proteins (7).

    Infection of pigtailed macaque PBMCs in vitro showed strikingly similar kinetics and replication patterns to those reported for human cells (see 610 Fig. 2A), and we observed extensive G-to-A hypermutation in vivo (see Fig. 5). It therefore might be expected that XMRV would be similarly hypermutated and restricted during any in vivo human infection, leading to comparably transient, low level viremia.'

    21. 'The potential importance of APOBEC-mediated hypermutation in limiting viral replication is underscored by the rapid decline of plasma viral loads to undetectable levels in our infected animals in the face of persistent vDNA positive cells in blood and LNs, demonstrating that a total elimination of infected cells, either by immunological or viral lytic mechanisms, was not responsible for the disappearance of viremia, but rather that these cells contained defective viral genomes that did not express viral gene products.

    In agreement with this notion, vDNA in LNMCs was first detected by X-SCA just after peak viremia and was still detectable at necropsy, however longitudinal LN sections were vRNA negative by ISH throughout the study.

    Although these vDNA positive cells in blood and LNs persisted at 119 dpi, innate immune responses in LN were only transiently detectable in the first week of infection, in stark contrast to the continual LN immune activation associated with chronic SIV infection (see Fig. 8), and cellular adaptive immune responses were negligible, consistent with the view that the hypermutated proviral genomes were rendered non-functional and did not express viral gene products.

    Taken together, these results suggest that APOBEC-mediated hypermutation is likely largely responsible for the limited viral replication we observed in our infected animals.

    22. 'These results may have broader implications concerning the potential for gammaretroviral infection of humans, where such concerns have been raised in particular regarding transfer of porcine gammaretroviruses to immunosuppressed humans in the setting of xenotransplantation (66).

    Because gammaretroviruses lack the accessory genes possessed by other retroviruses, such as HIV and SIV, they are ill-equipped to counteract intrinsic host restriction factors like the APOBEC3 proteins.

    23. 'As shown here, these primate host restriction factors can be remarkably effective at inhibiting gammaretroviral replication in vivo, and may explain, at least in part, why gammaretroviruses have not been significant pathogens in humans, including immunosuppressed individuals, despite close human association with their animal hosts.'

    24. 'Hypermutation of viral genomes also likely explains our unsuccessful efforts to rescue virus from differentially stimulated PBMCs collected at the studys conclusion using a LNCaP based reporter cell line (DERSE.LiG-puro) (data not shown). Given the extensive G-to-A hypermutation identified by X-SGS in cell associated vDNA (Fig. 4) it is likely that the proviral genomes were rendered incapable of producing infectious progeny.'

    Humans

    25. 'These results suggest that the isolation of replication-competent XMRV from human blood samples may be difficult or unlikely, particularly when working with samples from unknown post-infection time points that are not likely to represent peak viremia, and are in contrast to previous studies that reported an ability to isolate infectious XMRV from human PBMCs and plasmas similarly using LNCaP target cells (34, 40).

    While these disparities might be explained by methodological differences, it seems that XMRV would have to be less susceptible to human APOBEC3-mediated hypermutation in order for virus to be isolatable from PBMCs; however, previous work has shown that XMRV is highly susceptible to APOBEC3-mediated hypermutation in human cells (7, 19, 47).

    To successfully rescue virus from human plasma, particularly without the ability to select peak viremia time points, XMRV would likely have to replicate to higher sustained levels in humans than those observed in our pigtailed macaques or exist in human plasma in immune-complexes that have a less detrimental effect on viral infectivity.

    If the course of XMRV infection in pigtailed macaques does reflect the natural history of XMRV infection in humans, we would expect serological assays and PCR assays to detect vDNA in PBMCs to have the greatest likelihood of detecting XMRV infection in clinical samples, due to the greater stability of these parameters following XMRV infection in vivo.

    In contrast, we would expect assays to detect vRNA in plasma and assays to rescue culturable virus to be less likely to detect XMRV infection due to transient positivity and lack of sensitivity, respectively.

    One of the potential shortcomings of previous studies designed to identify XMRV infection in human samples using PCR, serology, immunohistochemistry, in situ hybridization, virus rescue, or other means has been the lack of bona fide in vivo derived positive control samples...

    ...Therefore, in addition to characterizing the natural history of XMRV infection in vivo, we initiated these studies in pigtailed macaques to generate and make available positive control samples for future analyses...

    The reagents we have generated here should facilitate future studies aimed at examining XMRV infection in in vivo derived samples (a). Though XMRV infection of pigtailed macaques doubtless does not perfectly model human XMRV infection, these results suggest that a small, physiological viral inoculum, in the setting of functional APOBEC proteins, will at best likely replicate only briefly and to extremely low levels, and might therefore be very unlikely to induce any significant pathogenesis.'
     
  15. natasa778

    natasa778 Senior Member

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    You are highlighting the bits that suit the agenda of 'no need to worry folks lets look the other way' brigade (which imo is highly driven by wanting to avoid the issue of contaminated biologicals, avoiding the V word).

    Again they don't account for very real and documented possibility of this suposed hypermutation by human APOBEC being very much dependent on you APOBEC polymorphism. In other words not everyone's APOBEC acts in the same way or has the same effect etc. Do I need to repost those papers once again? Have you even read them?

    What if there is a tiny minority of humans (say 1%) whose APOBEC is no match for your average gammaretrovirus. It is a no-brainer that medical science should err on the side of caution, and these attempts to write of MulVs as harmless to humans are scientists simply trying to cover their behinds. Or not doing their job properly. Ignorance or willful spin?
     
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  16. natasa778

    natasa778 Senior Member

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    Methinks that is exactly what the authors above are doing in the bits that you highlighted. They are leaving out some very pertinent facts and possibilities. ERV, who you love to quote, also does just that. Through ignorance or spin, take your pick.
     
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  17. barbc56

    barbc56 Senior Member

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    I would appreciate if you would repost these papers or point in their direction.

    Methinks, these are your opinions and driven by the fact that you want a retrovirus to work out. I don't understand how you can guess what the authors are doing or thinking just like it would be pure speculation for me to say what I said about your motives in the above sentence.

    What pertinent facts are left out?

    I understand you don't like ERV, but what does that have to do with the science behind this study? You can't judge scientiest just because the theories happen to be something you disagree with unless you can back them with facts.

    We need these facts and not sweeping generalizations, conspiracy theories and bias towards a retroviral theory to get to the bottom of this question. If the science was showing just the opposite, I would be defending it just as vehemently but it's not. I don't think it's too bold to say that at this point.
     
  18. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    Well here are a few references. There are plenty more if those who keep pushing APOBEC restriction could be bothered to look. (linked to peoplewithme)

     
  19. currer

    currer Senior Member

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    The striking omission of this paper is that it does not challenge the macaque's immune systems to test whether the vDNA latent in immune cells (the PBMCs and the lymph nodes) can replicate and produce active virions, as occurred in the previous macaque study.

    This is the role of immune tissue, so it is odd that the authors did not check whether, once infected with xmrv, it can work normally.
    A bit like taking your car to the garage, payng for a modification, and then NOT taking it out for a test run.

    The element of immune challenge in reactivating latent gammaretrovirus is an essential part of the infective hypothesis.

    This would tell them whether the hypermutation they rely on successfully blocked all replication with no escapes.

    I cannot understand why they omitted to do this.
    There is a lot of very specious reasoning going on in this paper and I smell FEAR.
     
  20. natasa778

    natasa778 Senior Member

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    It'll be quicker if you look up and read what I posted earlier.

    I'm not guessing. They spelled out what they are doing and thinking. I listed those facts, read my posts.

    The facts are provided, read my posts.
     

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