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Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice

Discussion in 'XMRV Research and Replication Studies' started by Jemal, Jul 10, 2012.

  1. Dusty Miller

    Dusty Miller

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    Dusty Miller said:
    Interesting questions. My understanding is that these simple retroviruses have no easy way to go latent. Unlike herpesviruses, for example, which have complex genetic machinery to regulate their active versus latent states, simple retroviruses like XMRV and MoMLV are always 'on'. They have strong enhancers and promoters of transcription that appear to be designed for maximum gene expression under all circumstances.

    However, it is true that retrovirus expression can be suppressed by the host cell after integration of the virus, and this might be considered a form of latency. I don't know whether experiments have been done to look at the fate of such integrated but inactive viruses in animals. Certainly, it would be difficult to obtain funding to explore these issues in primates, our closest relatives, for 30 years under many different conditions, given the fact that there is little evidence that these viruses are present or cause disease in humans. In contrast, a huge and well-funded effort to understand HIV, a known human pathogen, is underway. Obviously, researchers and funding agencies must make choices about where to focus their efforts for maximum benefit.​

    I don't agree with the statement that "MLV latency is easily established. Of course, it depends on your definition of latency. I just spoke with some other virologists here at my institute, and the definition that seems most reasonable is the persistence of a virus genome in the absence of virus production, but with the potential for production of fully-infectious virus at a later time. Herpes viruses do this quite well. The herpes virus genome can persist in neuronal cells for long periods without making infectious virus, but can 'reactivate' at a later time and produce fully-infectious virus. During this period of latency, some proteins are made by the virus genome, and these regulate the latent state. In this way, the virus can avoid detection by the immune system for prolonged periods, only to re-emerge later. Shingles, caused by herpes zoster, is a good example of such re-emergence from virus latency.

    Back to the question of MLV latency, the publications quoted above don't show that MLV latency is easily established.

    For http://www.ncbi.nlm.nih.gov/pubmed/3871491, the statement in the Abstract that "NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period" only means that it took 10 weeks for the lymphoma to be apparent. I couldn't get the full paper without paying an exorbitant fee, but it is likely that these mice were injected with virus at birth, such that the virus was continuously replicating during the 10-week period after injection and before the lymphoma became apparent.

    For http://www.ncbi.nlm.nih.gov/pubmed/187773, the statement in the Abstract that "These [data] suggested that MuLV (Scripps) could exist either as a productive persistent or nonproductive latent infection in C57BL/St mice" doesn't say which possibility the data support, and therefore, does not provide data that an MLV can indeed become latent.

    For http://www.nature.com/nature/journal/v283/n5745/abs/283404a0.html, the statement that "In such cases, lymphomagenesis seemed to operate through the agency of activated murine leukaemia viruses (MuLV) and occurred only after very long latent periods, and most (but not all) lymphomas were of host origin" again refers only to a "latent" period before the lymphoma was detected, and does not address whether the virus was latent or not. Same for the last paper cited above. Thus, the question of possible MLV latency has not been resolved by these reports.

    Regardless, what I think most people on this forum are interested in is whether humans can be infected by an MLV-like retrovirus, which is later undetectable in the blood but remains hidden in either an inactive "latent" state, only to re-emerge later and cause additional damage, or in a slowly replicating "persistent" state, where it may cause hidden damage. Such hidden damage might occur in the brain while the virus is undetectable in blood, and biopsy of the brain to find the virus is problematic. On the other hand, MLV-like viruses poorly infect nondividing cells, such as neural cells, and the data from XMRV-infected monkeys support this prediction (Onlamoon et al. Table 1, showing little if any infection of brain).
     
  2. Mark

    Mark Acting CEO

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    Commiserations Wally. Everyone hates it when that happens. I recommend the Lazarus plugin to make sure this never happens to you again, on any web site.
     
  3. currer

    currer Senior Member

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    I have got a question for Dr Miller.

    Autoimmune disease: A role for new anti-viral therapies?David H. Dreyfus
    http://www.sciencedirect.com/science/article/pii/S1568997211001753

    The paper Bob posted on this thread today http://forums.phoenixrising.me/inde...r-new-anti-viral-therapies.18620/#post-283021 pulls together a number of phenomena associated with recent research - Rituximab, viruses hiding in B cells EBV, expression of HERVS - even though it does not mention ME/CFS it could have much to add to this debate.

    Now what I would like to know is - why should this paper suggest that an autoimmune disease could be treated with retroviral integrase inhibitors? (These stop the integration of viral DNA into the infected cell)
    It says -"Other novel anti-viral therapies of chronic autoimmune diseases, such as retroviral integrase inhibitors, could be effective, although not without risk"

    If a HERV is expressed or activated -
    How much viral expression would there be and would this be similar to an an exogenous retroviral infection?
    What type of replication is it?
    Would you get virions produced and spreading to uninfected cells?

    Could this suggest one reason why some ME sufferers have improved on antiretrovirals?
    I remember reading a reference to MS being treated successfully by antiretrovirals
    http://multiple-sclerosis-research....9/case-report-is-ms-caused-by-retrovirus.html

    PS it is a nuisance that we have only got access to the Dreyfus abstract.
     
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  4. currer

    currer Senior Member

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    Quote "Regardless, what I think most people on this forum are interested in is whether humans can be infected by an MLV-like retrovirus, which is later undetectable in the blood but remains hidden in either an inactive "latent" state, only to re-emerge later and cause additional damage, or in a slowly replicating "persistent" state, where it may cause hidden damage. Such hidden damage might occur in the brain while the virus is undetectable in blood, and biopsy of the brain to find the virus is problematic. On the other hand, MLV-like viruses poorly infect nondividing cells, such as neural cells, and the data from XMRV-infected monkeys support this prediction (Onlamoon et al. Table 1, showing little if any infection of brain)".[/quote]

    A few biopsies have been done on nervous tissue (according to a pilot study of four autopsy cases by Donovan et al.) (Not sure if this is published.)

    One case showed marked dorsal root ganglionitis and the two other cases showed mild excess of lymphocytes with nodules of nageotte in dorsal root ganglia. The fourth case showed a vast excess of corpora amylacea in the spinal cord and brain of unknown significance, and no ganglionitis.

    Most ME sufferers disease, although lasting decades in some cases, is not progressive, and many stabilise, and some recover. If there is infection in the brain it must be slight enough to correspond with what we see happening to patients (i.e. we dont die, but continue with a prolonged illness).
    "Little, if any infection of the brain" is not necessarily reassuring in this instance. (unfortunately!)
     
  5. Dusty Miller

    Dusty Miller

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    The Knox et al. paper from the Jay Levy lab (http://www.ncbi.nlm.nih.gov/pubmed/21628393) goes a long way toward showing that no MLV-like retroviruses are present in CFS patients. They used an assay that is capable of detecting many different types of MLV in an attempt to detect such viruses in patient blood samples. They did this by two methods. First, they cultured peripheral blood mononuclear cells (PBMC; basically, all blood cells without the non-nucleated red blood cells) from patients with mink cells that are susceptible to many MLVs, removed the PBMC, and continued to culture the mink cells for 3 weeks to allow any viruses that might be present to spread. They then assayed for virus production from the mink cells by a variety of fairly non-specific assays for retroviruses. Second, they used the same non-specific assays for retroviruses to analyze medium from patient PBMC cultures that had been stimulated to grow by addition of PHA (phytohemagglutinin). This treatment can promote the replication of retroviruses present in the PBMC. In all cases the results were negative, indicating the absence of any MLV in blood cells from the patients.

    These results cannot prove the absence of all retroviruses in ME/CFS. Particular retroviruses may grow slowly making them difficult to detect, they might not infect some of the cell types used in the virus assays such as the mink cells, or the viruses may have moved from the blood into other tissues, a la Onlamoon et al. The assays used are capable of detecting XMRV, other xenotropic retroviruses, and the polytropic or MCF-type viruses that some have claimed to detect in CFS patients.
     
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  6. Mula

    Mula Senior Member

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    You are mistaking the terminology and the database in which there is no envelope gene sequenced for 22Rv1/CWR-R1 and there is no data in Paprotka where the XMRV virus was completely sequenced from 22Rv1 or CWR-R1. The gag-pro-pol from 22Rv1/CWR-R1 was assembled from the two cell lines. Fig S3 lists several primers used on the named cells, but they have assumed them to be complimentary to the VP62 virus, they have given no more proof that these primers are complimentary. It is necessary to revisit the cells individually in a lab where VP62 has been removed and see how many different proviruses are present.

     
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  7. Bob

    Bob

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    Oh, I hate it what that happens! What a nightmare! Bad luck Wally! :(
     
  8. Dusty Miller

    Dusty Miller

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    Bob, you are absolutely correct and are not mistaking the terminology!
     
  9. Mula

    Mula Senior Member

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    Herpes viruses do not come close the the ability of retroviruses to maintain latency and still cause disease, those references show MLVs in particular to have no difficulty. The latency in the NFS/N mice is the virus, nothing at all to do with the lymphoma. The paper from Jenson is from 1976 and Fathman is from 1980 and builds a history of research on MLV latency which was resolved by later papers. MLVs are commonly found infecting areas such as the spleen, thymus, bone marrow, which are easily accessible in comparison to the brain.

    [quoteThe role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3.[/quote]http://www.ncbi.nlm.nih.gov/pubmed/15254211
     
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  10. Mula

    Mula Senior Member

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    Bob has used the term region for what is putative envelope polyprotein when the question is why there is no envelope gene for this virus.
     
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  11. Bob

    Bob

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    What about Supplemental Fig. S3, A? Doesn't that show that full sequences were detected from each cell line?
    http://www.sciencemag.org/content/suppl/2011/05/31/science.1205292.DC1/1205292s.pdf

    How do you know that Mula? What is the evidence for that?
    The reasons that you gave me earlier were not adequate evidence for your claims, as far as I could work out, as I explained in detail earlier.

    Do you mean they haven't used the correct primers?
    Surely their primers can be checked against the VP-62 genome?

    Actually, I've just checked it myself, and if I use one of their env gene primers:
    8r 5’-CTGGATGCTACCGGAGCCC-3’
    And run a blast, it comes up with XMRV VP-62 100% identity.

    Same with their other env gene primer:
    F2r 5’-TTGCAAACAGCAAAAGGCTTTATTGGGAACACGGGT
    ACCCGGGCGACTCAGTCTATCGGATGACTGGC-3’

    That's also a VP-62 sequence.

    See the blast search here:
    http://blast.ncbi.nlm.nih.gov/Blast...Options_1SuC4f_1FSM_DTkZxcrQ3qq_23to07_21npY7

    I don't understand what you mean here Mula. Could you re-explain?
     
  12. Dusty Miller

    Dusty Miller

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    Sorry for taking so long to respond to your questions. I looked over the abstract of the Holmes papers you sent me. While the approaches he took to finding viruses are a good start, they are not very sensitive (the reverse transcriptase [RT] assay) and don't tell you what virus is present (electron microscopy [EM]). Today, I would follow up these findings with a sensitive assay for retroviruses, which if positive, allows one to identify the offending virus.

    For detection of retroviruses, I like the marker rescue assay. This is what I used to detect the retrovirus present in 22Rv1 cells, which turned out to be XMRV (http://www.ncbi.nlm.nih.gov/pubmed/19403664). For the marker rescue assay, one first generates a cell line containing a retroviral vector that carries an easily detectable marker gene. The retroviral vector is replication defective. For detection of viruses that are suspected of infecting humans, I use a human cell line carrying the retroviral vector. I also use a Mus dunni cell line that is broadly infectable by many different MLVs. Cells carrying the retroviral vector do not produce the vector, but infection of the cells with an MLV results in production of both the infecting MLV and the retroviral vector. Production of the retroviral vector is easily detected by infecting cells that don't express the vector or any other MLV, and then by staining the cells to detect transfer of the marker gene. A positive result is followed up by sequencing the new virus present in the cells, which can be done by a variety of relatively simple techniques.

    To detect retroviruses in blood, it's best to co-cultivate mononuclear blood cells (PBMC) with the marker rescue assay cells, which allows virus produced by the PBMC to directly infect the marker rescue assay cells. The cells are then passage to allow virus spread, and are assayed for vector production.

    This all may sound a bit complicated, but this assay can detect a single infectious virus particle produced by the patient blood cells. Once captured in this way, the virus is easily characterized. One can also assay for virus present in other materials such as cerebrospinal fluid, for example. If one suspected that the NZB virus was involved, as you propose in your questions above, it is easy to customize the assay to look for this virus. In the case of NZB virus infection, the marker virus produced would be able to infect human cells, but not lab mouse cells.

    My lab actually proposed to use this assay to detect possible MLV-like viruses in blood from CFS subjects, but we got bogged down by objections from bloggers on the mecfsforums site, and by our own Institutional Review Board (IRB), who were worried about the risk/benefit ratio of the proposed 30 milliliter blood draw! Several bloggers were worried that we might not find XMRV, while the IRB was worried about the possible impact of our findings on patients, regardless of the outcome. We finally gave up on the proposal.
     
  13. Navid

    Navid Senior Member

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    dear lord: i really hope the rantings of 5-10 somewhat unstable bloggers did not stop you from doing important research on a forgotten, scorned disease that robs so many of our lives. if it is true that their rantings stopped research into the disease it is truly a shame and a huge injustice to the millions of sufferers who are rationally, patiently, longlingly waiting for a treatment to regain their lives. most of us do not care if the cause of the disease is xmrv, abcd or sxyz.....you must know that the large, large majority of the people struck by this disease are ordinary people, who one day were raising their families, going to work or school, enjoying their lives and suddenly the next day were felled by a sinister life sucking disease. i hope in the future you will remember the lives of these millions, rather than become embroiled in silly debates with several loud outliers.....for the sake of of all the suffering sick and their families, i pray that if the opportunity arises again for you to look into causality and or treatment of ME/CFS you choose that path rather than the path of listening to the loud but certainly minority rabble rousing (at times incoherent)) crowd.

    all the rest of us want is our lives back but we need true scientific and medical research for that to ever become a reality.

    warmest regards
     
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  14. Dusty Miller

    Dusty Miller

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    No worries, the bloggers involved and my local IRB actually did us a favor, in spite of their intentions. My lab members and I, and Ecoclimber, who was helping us with the design of the trial, didn't know at the time that Jay Levy's group was essentially doing the study we wanted to do. Their study (http://www.ncbi.nlm.nih.gov/pubmed/21628393) was published before we could have completed our planned study, which would have made our study redundant.

    Anyway, thanks for your comments.
     
  15. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    I find this an interesting remark. By any definition, 'little if any' infection of the brain is infection of the brain. After all a little bit dead is dead. Considering the miniscule size of the study, it would be reasonable to assume there would be some with greater infection, and others with less in a larger study. Considering the time-frame of the study, and potential for 'latency' (as characterised by studies above, and not by word of mouth from fellow researchers), then your statement doesn't appear to hold water, at least to me.​
     
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  16. anciendaze

    anciendaze Senior Member

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    Dusty, I think consulting a specialist on DNA viruses about latency is disingenuous. Their DNA is in episomes outside chromosomes, thus requires independent regulation. Proviral DNA from retroviruses is integrated into chromosomes, which means other epigenetic controls are possible. Epigenetics is a fairly large subject.

    I'm still trying to imagine the mental model in play. Do you think ERVs corresponding to simple retroviruses (type I and II) are constantly transcribed? Do you think it is just a fluke that they can sit there quietly for millions of years? Do you think the ratio of proviral DNA to transcribed RNA in infection by simple retroviruses stays approximately constant? Everything I try to imagine seems ridiculous.
    I would hate to impute such reasoning to you, so there must be another explanation.
     
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  17. Dusty Miller

    Dusty Miller

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    Dusty Miller said:
    On the other hand, MLV-like viruses poorly infect nondividing cells, such as neural cells, and the data from XMRV-infected monkeys support this prediction (Onlamoon et al. Table 1, showing little if any infection of brain).
    More word games, what fun. So you think a dental X-ray is the same as looking into the melting reactor at Chernobyl? A drizzle is the same as a deluge? No shades of light in your world?

    What I was referring to is that only one of 5 animals studied showed infection of the brain.
     
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  18. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    Oh Dusty. Not much of a game for most of us here. Sorry you are under that impression, but it does perhaps explain why you are here. As for shades of light, I am extremely light sensitive as a result of my illness.:)

    I must have missed your reference to other negative studies, and for that matter, at least one other positive study (there may be more), in your post.

    My statement still stands, do you really think 5 studies showing no infection cancel one (more than one) that does? Please leave aside the flippant remarks about Chernobyl. I am sure those unfortunate patients who happen to be above the 'little if any' part of the curve might not view your response with a laugh.
     
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  19. jace

    jace Off the fence

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  20. acer2000

    acer2000 Senior Member

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    Dr Miller,

    Thanks for answering our questions. I am wondering if you can comment on the phenomenon that is described in this paper:

    Suppression of natural killer cell activity by Friend murine leukemia virus.

    http://www.ncbi.nlm.nih.gov/pubmed/6202923

    I have been curious about this ever since the link between XMRV (or really any retrovirus) and CFS was suggested. What is known about this? What is the mechanism and do other gamma-retroviruses (or retroviruses in general) share this ability to suppress NK cell activity? NK cell activity is known to be very low in people with CFS. Its one of the only markers that has been consistent across multiple research studies and appears to also follow the severity of symptoms. Secondary infections that are usually controlled by NK cells also are described as being more active in patients with CFS (ie herpes type viral reactivation) by most doctors who have experience in this illness. I'd be curious your take on this as it would seem that if we could figure out what is causing the shift in NK cell function, it might lead us to the offending pathogen.
     
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