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How Non-Cytolytic Enteroviruses May Spread From Cell to Cell

Discussion in 'Other Health News and Research' started by Hip, Sep 27, 2012.

  1. Hip

    Hip Senior Member

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    Non-Cytolytic Enterovirus May Play a Fundamental Role in ME/CFS

    In the chronic enterovirus infections found in ME/CFS, there is an unusual form of this virus called a non-cytolytic enterovirus. It is believed that this non-cytolytic enterovirus may be a major cause of ME/CFS.

    Unlike regular enteroviruses, non-cytolytic enteroviruses do not produce any new viral particles. An unlike regular enteroviruses, non-cytolytic enteroviruses live inside human cells as a long-term intracellular infection. Coxsackievirus B (CVB) and echovirus are two enteroviruses known to be able to create such chronic non-cytolytic infections.

    Non-cytolytic coxsackievirus B is found in the muscles and intestines of ME/CFS patients, the heart muscle in chronic CVB myocarditis, and is also found in the pancreas in type 1 diabetes, and in chronic inflammatory myopathy (an inflammatory muscle disease). Refs: 1, 2, 3, 4. This non-cytolytic CVB may be the major casual factor in these diseases.

    Coxsackievirus B or echovirus infection begins with the regular form of the virus that you catch. However, once inside the body, a regular enterovirus can transform into a non-cytolytic virus, and then take up long-term residence in your cells, forming a chronic intracellular infection that your immune system finds hard to clear.

    Prof Nora Chapman, Prof Steven Tracy, Dr John Chia and others have pioneered the understanding of chronic non-cytolytic enterovirus infections.



    Lytic and Non-Cytolytic Enterovirus

    In the life cycle of a regular (lytic) enterovirus, the virus enters a human cell, replicates itself thousands of times, then ruptures and kills the cell by lysis (lysis means cell rupture) so that the thousands of newly created viruses can escape, and go onto to infect more cells.

    By contrast, non-cytolytic enteroviruses live within human cells on a long term basis. They do not create any new viral particles, and they do not lyse the cell they live in (they do not kill the cell). They remain in the cell on a long term basis as a slow "smoldering" infections.

    Non-cytolytic viruses may nevertheless cause cellular dysfunction due to their presence in the cell, and provoke an immune response, which may lead to disease.

    Since non-cytolytic infections do not create any new viral particles, you might think that a non-cytolytic enterovirus infection would not spread to any new cells. However, a new study has discovered a mechanism by which non-cytolytic enteroviruses may spread from cell-to-cell in the body tissues.



    How Non-Cytolytic Enteroviruses May Spread

    This study demonstrates how non-cytolytic enteroviruses may be able to spread into adjacent cells. The study found that in CVB-infected cells, the virus seems to be able to create filament-like cellular protrusions that grow out of the cell, bridging to adjacent cells.

    Cellular protrusions are a normal part of cellular function: they are created by the cell when it wants to gain traction and pull itself along in the tissues — this is basically how cells can move.

    However, the authors suggest that these protrusions may be used by non-cytolytic CVB in order to transmit the infection into adjacent cells. In other words, non-cytolytic viruses may induce cellular protrusions to create a bridge to adjacent cells, which they then cross.

    There is a time-lapse video (supplemental file 1) in this study in which shows the creation and movement of these cellular protrusions produced in a non-cytolytic CVB infection:


    In the above video, cells infected with non-cytolytic Coxsackie B virus are shown on the left, and are seen sprouting the black thin filament-like cellular protrusions, which may carry the non-cytolytic infection cell to cell. The cells on the right are uninfected controls.

    Here is a snapshot from this video, showing the cellular protrusions (black filaments) running from cell to cell:

    cellular-protrusions.jpg
    Coxsackie B virus infected cells showing cellular protrusions
    (thin black filaments) running from cell to cell. These protrusions may
    transmit the non-cytolytic Coxsackie B virus from cell to cell.
    Uninfected cells do not display such protrusions.​



    More Info On Non-Cytolytic Enteroviruses

    Human Enteroviruses and Chronic Infectious Disease by Prof Steven Tracy and Prof Nora M. Chapman. I believe it was Prof Tracy et al at the University of Nebraska who first discovered non-cytolytic enteroviruses (in the heart muscle in coxsackievirus B myocarditis)

    Replication Defective Enterovirus Infections: Implications for Type I Diabetes by Prof Nora M. Chapman.

    Non-cytolytic enteroviruses are also called:
    • Non-cytopathic enteroviruses
    • Defective enteroviruses
    • Terminally-deleted enteroviruses
    Non-cytolytic enteroviruses may be detected by sensitive RT-PCR, and by immunohistochemistry (see page 22 of this document).



    Other Non-Cytolytic Viruses

    The enteroviruses coxsackievirus B and echovirus are known to produce non-cytolytic infections, but other viruses are also observed to create non-cytolytic infections.

    Dengue virus is known to produce non-cytolytic infections. And with dengue, you can get a post-infectious fatigue syndrome (see here), and this makes me wonder whether it is the non-cytolytic dengue virus which is causing this fatigue syndrome.

    Hepatitis B and hepatitis C are two others virus that produce defective viral infections, and again, chronic infections with these viruses often results in fatigue as a major symptom.

    Intriguingly, evidence for Epstein-Barr virus non-cytolytic infection has been found (see this study, which found defective EBV). So conceivably in EBV-associated ME/CFS, non-cytolytic EBV might be involved in the etiology.



    Non-cytolytic enteroviruses are analogous to the herpesvirus abortive infections that Dr Martin Lerner proposed are the cause of herpesvirus-associated ME/CFS.
     
    Last edited: Apr 17, 2018
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  2. Hip

    Hip Senior Member

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    One question that I have:

    Might it be possible to develop a drug that prevents the formation of these cellular protrusions in cells infected by non-cytopathic viruses?

    Such a drug would presumably halt the spread of the non-cytopathic enterovirus infection in the body, thus effectively eliminating this infection. Assuming non-cytopathic enterovirus infections play a pivotal role in ME/CFS, as has been suggested, then such as drug would also presumably cure ME/CFS.

    The only problem is that such a drug may well interfere with the normal functioning of the body, because cellular protrusions are in fact natural mechanisms that drive such processes as wound healing, immune responses, and cell migration. This is because cellular protrusions are the means by which cells pull themselves along within the body tissues.

    The cellular protrusions are akin to the arms and legs of the cell. The cellular protrusions themselves comprise a portion of the normal membrane of the cell that has been elongated to form this long tendril shape.

    Broad and flat cellular protrusions called lamellipodia sprout out in the intended direction of movement of the cell, adhering to surfaces ahead, thus allowing the cell to gain an anchor point and traction, and pull itself along in that direction. Quite amazing really. Long thin protrusions called filopodia are another important type of cellular protrusion. Filopodia act as the guiding "eyes" of the cell, with filopodia cellular protrusions extending ahead of the cell and exploring the immediate environment of the cell, sensing guiding cues, and directing the movement of the cell. Ref: 1
     
    Last edited: Jan 9, 2018
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  3. globalpilot

    globalpilot Senior Member

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    Hi,
    Just wondering a couple of things.
    a. if they are infecting other cells, does that not mean they are replicating ? If so, why would normal antiviral drugs (Ifor enterovirus) not be effective ? Dr Chia has had very good yet very short term success with ribavirin.

    b. I know that in order for a helper T cell response to occur , an APC in the blood must present the antigen. And the APC ingests the antigen in the blood. So I wonder if, by contining to live inside the cell and not enter the blood to reinfect, the immune response is not alerted to the infection ? If this is the case, is it possible a small vaccine would be effective in eliminating these viruses that are not infectious via the blood by alerting APCs to their presence ?

    GP
     
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  4. Hip

    Hip Senior Member

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    @globalpilot

    Non-cytolytic enteroviruses do replicate slowly, but they have very little cytopathic effect — in other words they do not kill the cells they inhabit. But they likely alter the functioning of the cells they infect, and they will elicit immune responses.

    Interferon (IFN) can destroy non-cytolytic enteroviruses: non-cytolytic enteroviruses are made from single stranded RNA (ssRNA) and double stranded RNA (dsRNA), and there is a natural interferon immune response that occurs when viral dsRNA is detected inside the cell. The interferon immune response to dsRNA is:

    viral dsRNA inside cell ➤ triggers TLR3 ➤ which releases IFN ➤ which releases RNase L

    The molecule RNase L then destroys the ssRNA inside the cell, both viral and cellular.

    Though dsRNA is resistant to destruction by RNase L

    And of course, as is well known, the RNase L molecules in ME/CFS patients are too small — low molecular weight RNAse L is one of the abnormalities of ME/CFS. So in ME/CFS RNase L does not function normally.


    Nora Chapman's presentation on enteroviruses and non-cytolytic enteroviruses contains the following slides:

    Slides from Prof Nora Chapman's presentation:
    "How Does a Lytic Enterovirus Persist and Cause Chronic Disease?"
    Terminally deleted CVB in mice.jpg
    In the above, TD viruses = terminally deleted coxsackievirus B3 = non-cytolytic coxsackievirus B3

    Enterovirus and CFS.jpg

    Note that the second slide above points out that lytic enteroviruses were not always found in chronic fatigue syndrome, in spite of the presence of enteroviral RNA.

    This fact suggests that non-cytolytic enteroviruses, rather than the normal lytic enteroviruses, are playing the major etiological role in ME/CFS.



    The Nature of Non-Cytolytic Enterovirus Infections

    Inside the capsid of an enterovirus, the viral genome consists of a single strand of positive sense RNA.

    RNA can exist in three forms: (1) single strand positive sense RNA, (2) single strand negative sense RNA, and (3) double stand RNA, which combines one positive single strand and one negative single strand together to make a double stand (like two halves of a zipper joined together).

    Normally in lytic enterovirus infections, a small number of negative-sense viral RNA strands, containing the full the viral genome, are created as templates. These templates are then used to make multiple copies of the positive-sense viral RNA strands; this is a bit like using a photographic film negative to make multiple photographic prints. These multiple copies of the positive-sense viral RNA strands are then packed into viral shell (capsids) as part of the virus replication cycle.

    In these lytic enterovirus infections of a cell, for every negative-sense viral RNA template (film negative), there are around 100 positive-sense viral RNA strands (photographic prints) made. So the ratio of positive to negative strands in the cell is around 100:1.

    However, in non-cytolytic enterovirus infections, it is found that there is roughly equal amounts of positive and negative strands in the cell, ie, the ratio is around 1:1.

    The positive stand RNA and negative strand RNA are thought to be able to intertwine together to make double stranded RNA (dsRNA). The immune system finds dsRNA more difficult to destroy. Dr Chia calls this dsRNA the "seeds", because dsRNA is tough and hardy, in the sense that dsRNA is hard for the immune system to wipe out.

    In the enterovirus infected muscle cells of ME/CFS patients, equal amounts of positive and negative RNA strands are found; so in ME/CFS the positive to negative ratio is 1:1.1 This indicates the presence of a non-cytolytic enterovirus infection in ME/CFS. And of course Dr John Chia's more recent research has demonstrated the presence of non-cytolytic enteroviruses in the stomach tissues of ME/CFS patients.1



    How Lytic Enteroviruses Turn Into Non-Cytolytic Enteroviruses

    When enterovirus enters a dividing cell, it creates a normal lytic infection, and very little non-cytolytic enterovirus is produced. However, when enterovirus enters a non-dividing, this is when it will tend to set up a non-cytolytic infection in that cell.

    Thus whether the cell infected is dividing or non-dividing, that is the crucial factor that determines whether a normal lytic enterovirus gets converted into a non-cytolytic enterovirus or not.

    In non-dividing cells, aka quiescent cells, (such as nerve, muscle, or heart muscle cells), enterovirus replication results in a mixed population of both lytic and non-cytolytic enteroviruses. So non-dividing, quiescent cells are where you can find non-cytolytic enteroviruses. Ref: page 21 of this document.

    More details of why non-dividing cells give rise to non-cytolytic infections are found in this post.



    More info on these non-cytolytic enteroviruses found in ME/CFS patients can be found in these two videoed presentations by Dr John Chia:
    Dr John Chia State of Knowledge Workshop on ME/CFS Research Part 1
    Dr John Chia State of Knowledge Workshop on ME CFS Research Part 2
    and in Dr Chia's seminal paper:
    The Role of Enterovirus in Chronic Fatigue Syndrome. 2005. J K S Chia

    The reason most antiviral drugs are ineffective against non-cytolytic enteroviruses is because antivirals work by one or more of the following mechanisms: (1) attachment or entry inhibitors, which prevent the viral capsid from attaching and entering the cell; (2) uncoating inhibitors, which prevent the virus from opening up its capsid and releasing its contents; (3) replication inhibitors, which prevent the virus from creating copies of itself inside the host cell; and (4) release inhibitors, which prevent the lytic release of replicated viruses from the host cell; however, non-cytolytic enteroviruses are not lytically released from cells, so many of these antiviral mechanisms will unfortunately have no effect against non-cytolytic enteroviruses.



    Summary Points on Non-Cytolytic Enteroviruses

    • A non-cytolytic enterovirus lives inside cells and consists of positive and negative strands of RNA, as well as double stranded RNA.

    • These positive and negative strands of RNA which are produced in equal numbers can intertwine together to make double stranded RNA (dsRNA). Dr Chia calls this dsRNA the "seeds" of the infection, because dsRNA is tough and hardy, in the sense that dsRNA is hard for the immune system to wipe out.

    • Non-cytolytic enteroviruses may be detected by sensitive RT-PCR, and by immunohistochemistry.

    • Non-cytolytic enterovirus infections tend to produce roughly equal levels of positive strand and negative strand RNA (in contrast to lytic enterovirus infections, which produce 100 times more positive than negative RNA strands).

    • Non-cytolytic enteroviruses are produced when enterovirus infects non-dividing, quiescent cells (like nerve or muscle cells). By contrast, when enterovirus infects dividing cells, very, very little non-cytolytic virus is produced.



    Google search for more info non-cytolytic enteroviruses.
     
    Last edited: Feb 2, 2018
  5. alex3619

    alex3619 Senior Member

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    Thanks for doing this investigation Hip. This is a topic I have wanted to look at more deeply for many months now, but never found the time to do.
     
  6. Hip

    Hip Senior Member

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    globalpilot

    I understand that the hypothesis that Nora Chapman et al have is indeed that the immune system is not alerted to non-cytolytic (non-cytopathic) enterovirus infections, as in these infections there is a low level of viral RNA and viral proteins being produced.

    Some details of the immune response to enteroviruses in general can be found HERE (see the immunity and immune response paragraph). Some excerpts:

    • T lymphocytes do not contribute to viral clearance and, in coxsackievirus B3 myocarditis, may contribute to myocardial inflammation.

    • Macrophage function is also a critical component of the immune response in enteroviral infections; ablation of macrophage function in experimental animals markedly enhances the severity of coxsackievirus B infections.
     
    Last edited: Apr 22, 2014
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  7. Hip

    Hip Senior Member

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    I have long been interested in trying to understand the enterovirus infection that I think likely underlies the bulk of ME/CFS cases.
     
    Last edited: Apr 22, 2014
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  8. cigana

    cigana Senior Member

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    Very interesting, thanks for making these posts.
    Do you know if the antiviral used successfully by Lerner (Valtrex I think) is effective against the non-cytopathic forms?
     
  9. Hip

    Hip Senior Member

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    Unfortunately I don't think Valtrex works against non-cytopathic enteroviruses, as far as I am aware.

    Intravenous interferon therapy is effective against non-cytopathic enteroviruses, and this has been used by Dr Chia. The drug Ampligen, which is an interferon inducer, is also effective against non-cytopathic enteroviruses. And the immunomodulator oxymatrine works as well.
     
    Last edited: Apr 17, 2018
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  10. frederic83

    frederic83 Senior Member

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    Chia found a lot of non-cytolytic virus in the stomach, small intestine and colon. Organs of the body that are not particulary rich in quiescent cells... Or maybe it infects the senescent cells inside those organs and the quiescent cells in others.
     
  11. Hip

    Hip Senior Member

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    I am not sure about the small intestine and colon, but in the stomach, it was the parietal cells that Dr Chia found to be infected with enterovirus. Parietal cells secrete hydrochloric acid and intrinsic factor.

    I just did a quick Google search, and apparently parietal cells are quiescent (terminally differentiated) cells:
     
  12. frederic83

    frederic83 Senior Member

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    By the way, that thread is very interesting, how this virus spreads via the protrusions is crazy. It is clear that the standard antivirals poorly work.
     
  13. Hip

    Hip Senior Member

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    Yes, it is pretty crazy. Although it is not known for sure that non-cytolytic enteroviruses spread this way, via these cellular protrusions.


    @halcyon recently pointed me to a Wikipedia article on virological synapses, which is another way of creating connecting tunnels from cell to cell. I am not sure of the exact differences between the cellular protrusions that enteroviruses may use to spread, and virological synapses, but they are a very similar concept.

    It seems that herpes simplex virus, HIV and HTLV may create virological synapses between cells, in order to transit themselves from cell to cell.
     
  14. Gemini

    Gemini Senior Member

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    Dr. Chia sent 30 samples to the CDC for testing (ref: Dr. Unger's CDC Conference Call, 2/23/15).

    Now a year later are there test results does anyone know?
     
  15. frederic83

    frederic83 Senior Member

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  16. Wishful

    Wishful Senior Member

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    If this kind of virus is the cause of ME/CFS, isn't that a readily testable hypothesis? Shouldn't the majority of patients have it? Is it simply too difficult to reliably find the virus in tissue?

    Also, does this hypothesis fit with abrupt temporary remissions? I've had those maybe a dozen times since developing ME, with a complete loss of symptoms over a period of minutes or hours, lasting for hours, followed by a return of symptoms over hours. It doesn't seem likely to me for non-cytolytic viruses hidden somewhere in a body.
     
  17. Hip

    Hip Senior Member

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    It's quite straightforward to detect non-cytolytic enterovirus in the tissues of ME/CFS patients.

    If you look at all the British studies conducted in the 1990s, non-cytolytic enterovirus was frequently found in the muscle tissues of ME/CFS patients. Dr John Chia later extended the British research, finding non-cytolytic enterovirus in the stomach of ME/CFS patients as well. Dr Chia now routinely tests the stomach tissues of his ME/CFS patients for enterovirus by biopsy.

    (Dr Chia chose to look for enterovirus in the stomach tissues rather than the muscles of ME/CFS patients because it is easier and less painful to take a stomach tissue biopsy than a muscle biopsy; the latter also leaves a scar.)

    So there is no doubt that non-cytolytic enterovirus is found in ME/CFS patients' tissues. But as you know, association does not automatically imply causation.

    However, further evidence that non-cytolytic enterovirus is a cause of ME/CFS comes from the studies like Chia's using interferon to fight off this enterovirus infection: you find that as interferon treatment reduces viral load, the ME/CFS symptom get better.
     
  18. Gemini

    Gemini Senior Member

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    Update: My notes from the 5/25/17 CDC Conference Call indicate when asked the status of testing of the Chia stomach tissue samples, Dr. Unger replied they had been tested and the results were "negative."

    Has anyone seen a published paper on this work?

    Be nice to know details about CDC's testing methods, samples, results, etc.[/QUOTE]
     
  19. Hip

    Hip Senior Member

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    No, but the CDC have a history of not really understanding what's going on when it comes to enterovirus testing in ME/CFS.
     
  20. perrier

    perrier Senior Member

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    [/QUOTE]
    Gemini
    What would 'negative' mean??
     

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