XMRV infection of Rhesus macaques

17 February 2011

rhesus macaqueThe first detailed study of infection of nonhuman primates with the retrovirus XMRV reveals that the virus establishes a persistent infection characterized by infection of multiple tissues. Viremia (virus in the blood) is low and transient, with proviral DNA detectable in blood lymphocytes. The results show that the Rhesus macaque can be used to study XMRV infection, transmission, vaccines, and antiviral drugs.

The subject of this study, the Rhesus macaque (Macaca mulatta), was selected because of its evolutionary proximity to humans and a comparable immune system. The monkeys used did not have antibodies to the capsid protein p30 of XMRV, indicating that they were not previously infected. Animals were inoculated intravenously with 3.6 million TCID50 of purified XMRV – a good amount of virus, to ensure infection. The virus used, VP62, was produced by transfecting cells with cloned viral DNA isolated from human prostate.

Virus in the plasma fraction of blood was assayed by quantitative RT-PCR. Of three animals infected, virus was detected in one animal at day 4 and not after day 14; and in a second animal from days 14-20. The third animal did not develop detectable viremia. Proviral DNA was found in peripheral blood mononuclear cells (PBMC) of all three monkeys for 3-4 weeks, indicating successful infection. At one month post-infection proviral DNA was no longer detected. Plasma virus was again detected in one of the positive animals on day 291, 16 days after being immunized with a mixture of XMRV proteins. This means that viral DNA had been present in this animal but was not detected. XMRV was detected in CD4+ and CD8+ T cells and NK cells, but not in B cells or monocytes.

Rhesus macaques infected with XMRV did not display obvious clinical symptoms. Analysis of peripheral blood revealed increases in the number of circulating B and NK cells. Anti-viral antibody titers were detected after infection and re-infection of animals but soon decreased.

Other infected animals were sacrificed during the acute phase of infection to identify pathological changes and sites of virus replication. No pathogenic consequences were observed except for the formation of germinal centers in spleen and lymphoid organs, changes that are expected after immune stimulation. Virus was detected in a wide variety of tissues, including spleen, lymph nodes, the lining of the gastrointestinal tract, prostate, testis, cervix, vagina, and pancreas, but not* in others including brain, heart, kidney, and bladder. Different types of cells were infected in different tissues: lymphocytes in lymphoid organs, macrophages in lung, epithelial or interstitial cells in other organs. The authors note that “this viral behavior appears specific to this virus”.

Here are some other comments and conclusions drawn from this study:

  • The authors suggest that in Rhesus macaques, XMRV causes first an acute infection, followed by a persistent chronic infection. A persistent infection lasts for long periods of time; a chronic infection is a persistent infection that is eventually cleared. Since the monkeys in this study were all sacrificed, it’s not possible to determine if the infection was cleared.
  • The presence of XMRV in certain blood cells resembles the pattern in a cohort of ME/CFS patients
  • Virus is present in the prostate early in acute infection – XMRV was identified in prostate tumors
  • The presence of XMRV in reproductive tract tissues is consistent with sexual transmission of infection
  • After the acute phase, virus levels are very low, but there could be a different outcome in individuals with immune dysfunction
  • One animal produced virus after immunization; perhaps immune activation results in cycles of virus production
  • The virus has an initial acute phase followed by reactivation. The authors comment: “While our study has not linked XMRV infection with pathogenic mechanisms that might lead to prostate cancer or chronic fatigue syndrome, we submit that such link, assuming it exists, would be a temporally distant one.”
  • It would be informative to determine if XMRV is present in some of the same tissues in humans that were observed to be infected in rhesus macaques

Because the study involved only a small number of monkeys (8), the experiments should be repeated with additional animals, and in different laboratories, to verify the findings. I also wonder if the choice of the intravenous inoculation route had an effect on the pattern of infection and tropism. It is well known that viral pathogenesis can be determined by how the virus enters the host. For example, the same virus may replicate in different tissues, or have different virulence, when inoculated in different ways. This question can be readily addressed by inoculating rhesus macaques via different routes.

Studying viral pathogenesis (the series of events that occur during viral infection of a host) in animals is essential for understanding how viruses cause disease in humans. However, the results of such studies must always be interpreted with caution, because what is true in an animal is not always true for a human. For example, simple differences in size, metabolism, and development can have substantial effects on pathogenesis. In interpreting the results of animal studies, we must keep in mind the adage, ‘Mice lie, monkeys exaggerate‘.

Update: *These are the results of immunohistochemistry (IHC), which detects viral proteins and likely the produce of viral replication. When the IHC-negative tissues were examined for the presence of viral nucleic acids, low frequency signals were detected. The authors speculate that this is likely a consequence of failure of XMRV to replicate in these tissues.

Onlamoon, N, DasGupta, J, Sharma, P, Rogers, K, Suppiah, S, Rhea, J, Molinaro, RJ, Gaughan, C, Dong, B, Klein, E, Qui, X, Devare, S, Schochetman, G, Hackett, J, Silverman, R, & Villinger, F (2011). Infection, viral dissemination and antibody responses of Rhesus macaques exposed to the human gammaretrovirus XMRV Journal of Virology

  • http://www.facebook.com/drspeedy.andme Drspeedy Andme

    Thanks for explaining that Professor.

  • Anonymous

    Thanks for this, Prof. R. Any chance you could also offer commentary on this simultaneous publication?

    http://jvi.asm.org/cgi/content/abstract/JVI.00046-11v1

    Severe Restriction of Xenotropic Murine Leukemia Virus-Related Virus Replication and Spread in Cultured Human Peripheral Blood Mononuclear Cells

    Chawaree Chaipan, Kari A. Dilley, Tobias Paprotka, Krista A. Delviks-Frankenberry, Narasimhan J. Venkatachari, Wei-Shau Hu, and Vinay K. Pathak*

  • Kati

    Vincent, thank you for your analysis.

    You wrote: “Because the study involved only a small number of monkeys (8), the experiments should be repeated with additional animals, and in different laboratories, to verify the findings”

    It sounded that the costs were quite prohibitive at 50 000$ a monkey.
    However I believe a following article mentioned a mouse model for XMRV, with a very specific type of lab mouse.

    Other than cost, could you comment on the difference of animal models for this type of disease?

    Thank you!

  • just asking?

    why don’t the scientist use xmrv+ patients who are sick w/me and prostate cancer and do tissue and bone marrow biopsies, also measure t-cells and inflammation levels…just asking?

    if patients volunteer to be used in this way is against the law?

    if you want to see what xmrv is doing ito sick people, why don’t you use XMRV+ people who are ill and look at what the RV is doing to their blood, tissues, bone marrow, etc?

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  • #3622

    Does this research have any implications for that claims of Greg Towers & the Wellcome Trust that XMRV could not be circulating in the human population? Could a contaminant affect monkeys in this way?

    Also… weren’t these results released ages ago? Is this a different study, or did it just take a long time to get published?

  • http://twitter.com/smashman42 Ashley Hinds

    Do Rhesus macaques have an equivalent of the Herpes virus family (especially HHV3 -6) that are commonly found in abnormal levels in ME/CFS patients?

    Just wondering if co-infecting an animal model with say the equivalent of EBV might bring out something interesting.

  • Gob

    Wellcome Trust and Towers press release was wrong anyway

  • http://pulse.yahoo.com/_PNBRQBVFNNYBTSVPSSEQYSEO5I Tracy

    Thank you for the detailed explaination on the materials and methods of the study. The other blogs were sketchy on details. You are my new favorite virologist:)

  • Anonymous

    Thanks for covering this. It seems to be that they should set up a study where they look in the same organs in PC and CFS patients. Perhaps this explains why its hard to find in the blood?

    Also, its possible that XMRV might not directly cause symptoms, but might prevent the body from recovering from other infections when the immune system activates in in response to them years later. Remember, HIV itself doesn’t kill AIDS patients, but it sets the stage for them not to recover from other infections.

    The monkey study states that they were able to re-activate XMRV in the blood by stimulating the immune system. I think that they should do a study of CFS patients where they use the same tactic and see if they can detect it. Or perhaps because CFS patients feel worse after exercise, they should do a study where they test for XMRV before and after exercise.

  • Anonymous

    3622: This research was presented at the 1st International Workshop on XMRV at the NIH in September, but it had not yet been published at that time.

  • http://www.virology.ws profvrr

    As you know, mice and monkeys are very different. Mice are convenient
    to use in virology, at much less cost than monkeys, and they are
    genetically manipulable. There is certainly a place in XMRV research
    for mice – for example, to explore an antiviral or vaccine strategy
    before going into nonhuman primates. This is usually the course of
    action when studying a virus. I’m sure we will see more work on XMRV
    in mice – few groups have the money to use nonhuman primates. But
    nonhuman primates are more likely to produce results relevant to
    humans than mice, especially in terms of how a virus causes disease.

  • http://www.virology.ws profvrr

    In fact this work should stimulate research into understanding where
    XMRV can be found in humans. It is not against the law to study the
    sites of virus replication in humans, so long as proper Institutional
    Review Board approval is obtained. Before this study, it wasn’t clear
    where XMRV should be looked for in humans.

  • http://www.virology.ws profvrr

    The authors of this papers noted the precautions they took to ensure
    that their assays were not compromised by contamination. The rhesus
    monkeys were not previously infected with XMRV, and their reagents
    were free of mouse DNA contamination. These studies have been reported
    at meetings, but now they have been peer-reviewed and published in a
    journal. I try to write only about peer reviewed research.

  • http://www.virology.ws profvrr

    Many rhesus macaques in colonies are infected with rhesus
    rhadinovirus, RRV-1 and RRV-2. These are similar to the human Kaposi’s
    sarcoma virus, or HHV-8. These viruses latently infect the monkeys and
    are activated to replicate when monkeys are infected or immunized. The
    extent to which RRVs influenced the results in this paper are not
    known.

  • http://www.virology.ws profvrr

    You are right, from the results of this study it is clear that we
    should be looking for XMRV in other organs, rather than blood. As the
    authors write, if XMRV is involved in CFS and PC, the infection
    probably occurred some time before disease. And the findings do
    suggest why the virus is difficult to detect in the blood. Even
    proviral DNA was not detected, although in at least one monkey it was
    still present.

  • drosha

    Even though this research is encouraging for understanding this virus, my biggest problem with XMRV as a causative agent of any disease is that I’m not aware of any virus in humans that causes an apparent disease without any cytopathology. From what I understand so far this virus doesn’t cause cell death.

  • sue

    not sure why they did not test inflammatory markers in the monkeys..

  • Zac

    Nice picture. Thanks for the observations.

    Mice lie. monkeys exaggerate. I guess since this is essentially a mouse virus in a monkey, then the truth maybe lies somewhere in the middle. I’ll listen to TWIV 55 in a little while.

  • http://www.facebook.com/people/Connor-Bamford/652096932 Connor Bamford

    Hi Vincent, You wrote|: “It is well known that viral pathogenesis can be determined by how the virus enters the host. For example, the same virus may replicate in different tissues, or have different virulence, when inoculated in different ways.”

    I was wondering what is the current understanding of how XMRV may enter humans? Is the evidence suggestive of blood-borne or respiratory inoculation?

    What are the major factors then influencing differential pathogenesis? Could it be the specific immune nature of the site, i.e the presence of dendritic cells in the lower respiratory tract?

    Very interesting study – thanks for explaining!

  • Andy Millman

    > In interpreting the results of animal studies, we must keep in mind the adage, ‘Mice lie, monkeys exaggerate‘.

    Vincent,
    why do we need adages to help us interpret the data? Why can’t we just rely on the science? We have a potentially new human infectious retrovirus on the scene. Surely the situation requires a bit more gravitas than quoting glib adages?

  • http://www.virology.ws profvrr

    Nearly every scientist who works with animals is aware of that adage.
    It’s a good way of summarizing the problems with animal studies. In no
    way am I being glib. if you know anything about what I’m trying to do
    at this blog, you would know otherwise. The post is all about science,
    by the way.

  • Flex

    Vincent if mice lie and monkeys exaggerate, which negative study was run by mice and why are psychiatrists now looking like monkeys?

  • http://pulse.yahoo.com/_OBL2JO6QOAT3MY5PAD4U5G5QCI Ben

    An interesting blog article Professor, thank you for sharing this with us.

    In reply to a comment by ‘drosha’ concering lack of evidence for PCD and XMRV it should be noted:

    PBMC Apoptosis – Programmed Cell death- has been demonstrated in *both* CFS children (1) and adult groups (2) by researchers in Scotland, UK.

    This would indicate a possible active infection, tie this in with MULV/XMRV and things get interesting as the CFS XMRV studies are in PBMC’S.

    Mitochodrial PBMC ATP in CFS is also very low. (3).

    Kind Regards.

    References:
    ————–
    1): Biochemical and Vascular Aspects of Pediatric Chronic Fatigue Syndrome
    Gwen Kennedy; Faisel Khan; Alexander Hill; Christine Underwood; Jill J. F. Belch
    Arch Pediatr Adolesc Med. 2010;164(9):817-823.

    2): Increased neutrophil apoptosis in Chronic Fatigue Syndrome. Kennedy G et al. J Clin Pathol 2004:57(8):891-893)

    3): Chronic fatigue syndrome and mitochondrial dysfunction, Int J Clin Exp Med (2009) 2, 1-16 . Myhill S, Booth NE, McLaren-Howard J.

  • Patricia

    As an additional comment to that, Hepatitis C virus doesn’t cause “visible” cytopathology, especially not on a cellular level; for example, in cell culture it is impossible to tell the difference between infected and uninfected cells without staining for viral proteins. The virus is “non-lytic” – it leaves the cell without lysing or bursting the cell, so the cell can continue to produce virus for great lengths of time without dying. On an organ-wide scale, infection does not cause visible damage until sometimes years after initiation – and I doubt that there have been a lot of random organ biopsies of CFS patients looking for cyto/histopathology. (I could be wrong about that.)
    I hope this is what you were referring to, Drosha (also I enjoy your name, as our lab works on components of the microRNA biogenesis pathway – and Drosha is one of the major players :D).

  • http://www.virology.ws profvrr

    Many viruses are noncytopathic in cell culture, yet cause disease in
    humans. The tissue/organ damage is believed to be a consequence of
    immunopathology – the immune response damages cells and tissues. There
    is experimental support for this mechanism of pathogenesis for a
    number of human viral diseases. See
    http://www.virology.ws/2009/01/23/immunopathology-too-much-of-a-good-thing/.

  • Omerbasket

    Prof. Racaniello, the sentence about virus not detectable at tissues “including brain, heart, kidney, and bladder” is incorrect, since this is what it says in the study (and there are pictures in figure 8 to prove it…):
    “Other organs scored negative
    for XMRV by IHC (Table 1, brain, heart, kidney, bladder, gall bladder, etc.). These tissues were
    however not clear of XMRV since low frequency nucleic acid signals were detected by FISH
    compared to other organs, suggesting generalized dissemination of XMRV (Figure 8r-u), but
    lack of replication due perhaps to a paucity of suitable target cells, an unsuitable environment or
    some form of active control of viral replication which remains to be determined.”.

  • http://www.virology.ws profvrr

    A positive signal by immunohistochemistry indicates viral proteins,
    clearly viral replication. The in situ signal was much weaker and
    might not represent bona fide replication. I will clarify the post.

  • sue

    i am very skeptical about whether the findings regarding the brain can be extrapolated to humans. i had evidence of brain infection right from onset, complete with neck pain, fog, and inflammation. it is possible that this could develop from cytokines also, but i dont think thats the case because it is so similar to HIV brain infection symptoms.

  • drosha

    in order to tie in that data with XMRV, you need to show that 1. those patients have XMRV in their PBMCs, 2. XMRV causes cell death in human PBMCs or any other cell for that matter and finally 3. you need to find the mechanism with which this virus can cause cell death.

  • just asking?

    thanks Vincent……so we patients may now be able to hope that these type of studies will soon start on people….now that the monkeys have given us some leads where to look? if so, yay!!

    i ask /think abt this because I hate to see poor monkeys killed when there are so many of us sick and willing to be used to find the anwers….gives me both hope and purpose in life.

    thanks to the monkeys who died and or suffered so that i might some day have my health back : )

    have a good weekend vincent.

  • Danylle

    But all the negative Retrovirolgy papers weren’t peer reviewed right?

  • http://www.virology.ws profvrr

    They were peer-reviewed. See http://www.retrovirology.com/ “A peer
    reviewed journal that publishes reviews and original research on
    retroviruses.”

  • drosha

    Good point about HCV (also good point by Vincent). I guess I should change my hypothesis in this case to: I don’t know any disease causing human virus that doesn’t lead to direct or indirect cell death. It seems like the research indicates mostly indirect cell death associated with HCV infection.
    Drosha is not my real name but I use it as a screen name because it is my favourite gene name :)

  • Megan

    Thanks for you great blogs Dr Racaniello,

    I am also interested in the following comment you made :

    “I also wonder if the choice of the intravenous inoculation route had an effect on the pattern of infection and tropism. It is well known that viral pathogenesis can be determined by how the virus enters the host. For example, the same virus may replicate in different tissues, or have different virulence, when inoculated in different ways. ”

    My question here is with the ‘second hit’ XMRV ME/CFS theory in mind – could the pathogenisis of XMRV also vary based on the nature of a secondary illness that infects someone?

    eg. if a healthy person was carrying low levels of XMRV and was subsequently infected by either EBV or say an enterovirus and if these triggered further XMRV replication, then could the pathogenisis or pattern of infection vary based on the seconary illness?

  • Danylle

    I think the Editor can just approve them for publication according to this- from below, The Associate Editor can call upon an outside expert should s/he feel the need :

    Peer review policies

    A manuscript submitted to Retrovirology will be evaluated by the Editor-in-Chief, who may decline it or assign it to an Associate Editor for review. The Associate Editor then recruits one or more of the Editorial Board to give comments, and based on these s/he will reject, accept or request revisions of the manuscript. The Associate Editor can call upon an outside expert should s/he feel the need. It is expected that the review period will not exceed three weeks. Based on the reviews, the Associate Editor will make a recommendation to the Editor-in-Chief for rejection, revision or acceptance. Rejected works are permitted one additional round of re-submission. If the resubmitted work is declined again the decision is final.

    Edited by Kuan-Teh Jeang, Retrovirology is supported by an expert Editorial Board.

  • http://www.virology.ws profvrr

    That statement does not mean that the papers are not reviewed – they
    are reviewed by members of the Retrovirology editorial board, who are
    research scientists.

  • Danylle

    Oh yes sorryI read it again. It appears that it’s possible that all that would be necessary is for 2 researchers to look papers for publications. Is that about standard for peer review. I wonder how many researchers are required on papers from Science.

  • Hip

    It would be a very good idea to extent this excellent experiment, and after introducing XMRV, inoculate these animals with a second virus, choosing a virus statistically associated with CFS, such coxsackievirus B1 and B4, HHV-6A, EBV, parvovirus B19.

    Since many people with CFS note their disease started after a respiratory virus infection, it seems likely that CFS will only appear when you say XMRV plus one (or more) of the above respiratory virus.

    XMRV may be similar to HIV, in that it is the retrovirus which causes the immunosuppression, but the secondary infections are generally what cause the symptoms AIDS.

    So it may well be that you don’t see CFS symptoms until there are enough secondary infections in the body.

    Most people with CFS will be seropositive for say EBV, HHV-6, perhaps herpes simplex 1, perhaps cytomegalovirus, quite likely one or two Coxsackie B viruses, sometimes more.

    In addition, they will have a few species of semi-pathogenic bacteria in their system, like Staphylococcus, Chlamydia pneumoniae, Klebsiella, etc (which can express some nasty toxins).

    So inoculating more CFS viruses into these already XMRV positive animals, to note when and how CFS develops would be a very worthwhile experiment. Often it can take a few years from the initial respiratory infection before CFS starts to fully settle in, though people often notice significant CFS symptoms just months or even weeks after they first acquired their respiratory virus.

  • USAman

    There is one guy who can sort this mess out, its Dr John Coffin,
    he is the best we have got.
    Dr Coffin – Please help end the suffering of ME CFS sufferers. We believe in you.

  • Tiggerkenwood

    If this work should stimilate research into understanding where XMRV can be found in humans, then do you know why WPI having trouble getting permission to study tissue in XMRV patients for the XMRV studies from the same review boards you mention?

  • Anonymous

    Another question, if you don’t mind, Prof R –

    “Rhesus macaques infected with XMRV did not display obvious clinical symptoms. ”

    I have listened to a number of your excellent Virology 101 lectures, and it seems to me there are many examples given of viruses that infect animal hosts without causing disease, but which cause disease when they move into human hosts.

    If, hypothetically, XMRV causes a given disease in humans, is there any basis to assume that it necessarily would cause the same or a similar disease in macaques, even if the parts of the body that are infected turn out to be the same or similar?

    (p.s. Eight monkeys at $50,000 per monkey = $400,000. That’s equivalent to 7 percent of the ENTIRE NIH budget [$6 million] for biomedical research into ME/CFS for FY 2011…without taking into account any of the other costs of the study. Sometimes numbers make my head hurt.)

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  • http://www.facebook.com/profile.php?id=100001439936317 Lionel Berthoux English

    test

  • Kathryn

    Posted by Anonymous on Friday, February 25, 2011 4:08LikeDislikeReport SPAM
    Do you know about Ila Singh’s autopsy study? Her Patent lists the organs in which she found XMRV:

    (WO/2010/132886) COMPOSITIONS AND METHODS RELATING TO XMRV-RELATED DISEASES AND CONDITIONS

  • Mcharles

    I have been ill with cfs for 21 years . It was like the worst flu I ever got and I never recovered. This rhesus macaques study is very interesting.I tested positive for XMRV in April 2010.I looked back on my blood work from Dr. Cheney in 1992 and sure enough my cd4 and my cd8 and my nk cells were abnormal. My neck and face has been chronically swollen which Dr. Cheney told me that was lymphatic congestion. I developed a quarter size lump on my right testicle which went away in two months. Please keep research progressing in this area. I believe you are on the right track with XMRV. Thanks, Mike

  • Sduffy

    Professor in case you haven’t seen it I just wanted to give you a heads up that Kent Heckenlively at http://www.ageofautism.com/2011/02/immunization-provokes-xmrv-reactivation-in-monkey-model.html is presenting your study with his usual anti-vaccine slant.

  • http://www.virology.ws profvrr

    Every virus is different, so we should not assume that what we find in
    monkeys with XMRV is the same as in humans. With sufficient
    experimentation, and comparison of results in humans and monkeys, one
    could eventually determine if the disease is similar in both hosts.
    There are many examples of viruses that have very different outcomes
    in humans and in nonhuman primates. For example, Old World monkeys
    cannot be infected with HIV-1.

  • USAman

    Dr Vincent,
    Please can you help explain,
    Dr Greg Towers has a new paper out and says he believes prostate cancer is contamination, he says he found Identical integration sites that have never previously been described in any retrovirus infection.
    However does the fact the Rhesus macaque got infected and that they showed signs of a real infection, mean that Dr Towers findings don’t stop this being a REAL virus??

  • XMRV + since 2010

    Brain Demyelination/lesions AIDS Yes ME/CFS Yes

    Chronic sore throat Flu like illness AIDS yes ME/CFS yes

    Swollen lymph nodes AIDS yes ME/CFS Yes

    Cognitive problems AIDS yes ME/CFS yes

    Skin Problems AIDS yes ME/CFS yes

    IBS and other stomach problems AIDS yes ME/CFS Yes

    Cancer/Leukimia/Lymphoma AIDS yes ME/CFS Yes

    Weird tumor
    spleen,liver,brain,testicular AIDS yes ME/CFS yes

    Heart Failure AIDS yes ME/CFS yes

    Many active viruses AIDS yes ME/CFS yes

    White thrush AIDS yes ME/CFS yes

    Swollen tongue with teethmarks around it AIDS yes ME/CFS yes

    Fatigue AIDS yes ME/CFS yes

    Seizures AIDS yes ME/CFS yes

    How blind and gullible do you have to be, to not be able to see that ME/CFS is also caused by an HIV like virus? ME/CFS is caused by a retrovirus, we have known this since the early 80s when ME/CFS brain MRIs were taken and they looked exactly like AIDS patients, in the early 90s De Freitas found a retrovirus in the blood of ME/CFS patients and the CDC didn’t even bother paying attention to her, now the WPI has found the XMRV virus which is a retrovirus which was suspected all along and people still can’t admit that ME/CFS is caused by just that.,

    How blind and dumb are you?

    How can you tell me that is all in my head ?when two of my friends that have HIV have the very same health problems that I do but they do much much better then me because they have meds and I don’t.