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How Non-Cytopathic Enteroviruses May Spread

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

  1. Hip

    Hip Senior Member

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

    Dr John Chia, Prof Nora Chapman, Prof Steven Tracy and others think that, in addition to normal lytic enteroviruses, non-cytolytic enteroviruses (aka: non-cytopathic enteroviruses) may also be playing a fundamental role in ME/CFS.

    A normal enterovirus infection begins with the lytic form of the virus that you catch. However, once inside your body, lytic enteroviruses can convert into non-cytolytic enteroviruses, and the latter are very different entities, with very different life cycles.

    Non-cytolytic enteroviruses have been associated with ME/CFS, as well as a number of other diseases such as type 1 diabetes, myocarditis, cardiomyopathy (in a murine model), and chronic inflammatory myopathy. Non-cytolytic enteroviruses may conceivably be the primary casual agent in these diseases.


    Difference Between Lytic and Non-Cytolytic Enteroviruses

    As its name suggests, in the life cycle of a regular lytic enterovirus, the virus enters a human cell, replicates itself thousands of times inside the cell, and then ruptures and kill the cell (lysis) so that these thousands of replicated viruses can escape the cell, and go onto to infect more cells.

    By contrast, non-cytolytic enteroviruses live within human cells on a long term basis, and do not lyse (do not rupture and kill) the cell they live in, and do not burst out of the cell as lytic viruses do. And even they were able to burst out of the cell, these non-cytolytic viruses would not survive for long outside the cell, because non-cytolytic enteroviruses do not have an outer shell (capsid) to protect them.

    So you might think that a non-cytolytic enterovirus infection would not spread any further than the cells it lives in. However, a new study uncovers a mechanism by which non-cytolytic enteroviruses may spread in the body tissues.


    How Non-Cytolytic Enteroviruses Might Spread in the Body Tissues

    The following study reveals a mechanism by which non-cytolytic enteroviruses may be able to transmit copies of themselves into adjacent cells, thus facilitating their spread to and infection of more cells:

    Coxsackievirus B3-Induced Cellular Protrusions: Structural Characteristics and Functional Competence

    This study shows that in coxsackievirus B infected cells, this virus seems to be able to induce cellular protrusions to grow out of the cell. Cellular protrusions are tentacle-like filaments that issue from the cell, and are a normal part of cellular function. Cellular protrusions are used by the cell when it wants to gain traction and pull itself along in the tissue spaces. However, the authors posit that these protrusions may be the means by which non-cytolytic coxsackievirus B infections are able to transmit into adjacent cells: the authors suggest that non-cytolytic viruses may induce these cellular protrusions to create a bridge to adjacent cells, which they then cross, in order to infect the nearby cells.

    So even though non-cytolytic viruses do not have a protective outer shell, they may still be able to slowly spread to and infect more cells.

    There is actually a time-lapse video attached to this study in which you can watch the putative spread of non-cytolytic coxsackievirus B by means of the cellular protrusions it induces; this video can be downloaded from here (see supplemental file 1). In the video, the infected cells shown on the left hand side of the picture are seen sprouting the cellular protrusions that may be spreading the non-cytolytic viral infection from cell to cell. The cells shown on the right hand side are uninfected cells, used as a control.

    Here is a snapshot of this video, showing the tentacle-like cellular protrusions running from cell to cell:

    Cellular-Protrusions.jpg
    Coxsackie B virus infected cells showing cellular protrusions
    (thin filaments) running from cell to cell. These protrusions may
    transmit the non-cytolytic Coxsackie B virus to adjacent cells.​


    More Information About Non-Cytolytic Enteroviruses

    Human Enteroviruses and Chronic Infectious Disease. Steven Tracy and Nora M. Chapman.
    Replication Defective Enterovirus Infections: Implications for Type I Diabetes. Nora M. Chapman.

    Lytic enteroviruses are also called: cytolytic enteroviruses, cytopathic enteroviruses, or wild-type viruses.

    Non-cytolytic enteroviruses are also called: non-cytopathic enteroviruses, defective enteroviruses, and terminally-deleted enteroviruses.

    Non-cytolytic enteroviruses may be detected by sensitive RT-PCR, and by immunohistochemistry (ref: see page 22 of this document).
    Last edited: Apr 23, 2014
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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.

    Under normal circumstances, cellular protrusions are the everyday means used by cells to move 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.
    Last edited: Apr 22, 2014
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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
  4. Hip

    Hip Senior Member

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    globalpilot

    I understand that non-cytolytic enteroviruses do replicate very slowly, but they have very little cytopathic effect — they do not kill the cells they infect and inhabit. But they likely alter the function of the cells they inhabit.

    Interferon (IFN) can destroy non-cytolytic enteroviruses: non-cytolytic enteroviruses are made from naked 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 viral RNA inside the cell, both viral and cellular.

    Though the dsRNA is more resistant to destruction by RNase L than other forms of RNA. 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 this RNase L does not function as efficiently as it should in destroying viral RNA and dsRNA in the cell.


    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 (aka: cytopathic 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 regular cytopathic (lytic cycle) 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).

    Positive-sense viral RNA is the same format as human messenger RNA (mRNA) used by cells for gene expression, and thus a positive-sense RNA virus has the advantage that its genome can be immediately translated by the host cell into proteins to build new virus particles.

    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 (the photographic film negative), there are around 100 positive-sense viral RNA strands (photographic prints) made. In other words, the ratio of positive to negative strands in the cell is around 100:1.

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

    These positive to negative strands then intertwine together to make double stranded RNA (dsRNA), and this double stranded RNA is in fact the non-cytolytic enterovirus.

    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. 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.


    How Lytic Enteroviruses Turn Into Non-Cytolytic Enteroviruses

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

    When dividing cells (such as those in the liver) are infected by enterovirus, viral replication processes in these cells produce lytic enteroviruses almost exclusively, with only a tiny quantity of non-cytolytic enteroviruses appearing. So non-cytolytic enterovirus infections are not really found in dividing cells.

    However, when non-dividing cells, aka quiescent cells, (such as nerve, muscle, or heart muscle cells) are infected by enterovirus, viral replication processes in these quiescent cells produce 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 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 do not have capsids, and are not lytically released from cells, so most of these antiviral mechanisms will unfortunately have no effect against non-cytolytic enteroviruses.



    Summary Points on Non-Cytolytic Enteroviruses

    • A non-cytolytic enterovirus consists of a naked strand of dsRNA that lives inside cells

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

    • Non-cytolytic enterovirus infections produce nearly 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).

    • These positive and negative strands of RNA which are produced in equal numbers intertwine together to make double stranded RNA (dsRNA). This double stranded RNA is the non-cytolytic enterovirus.

    • 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.
    Last edited: Apr 22, 2014
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  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.
    Last edited: Apr 22, 2014

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