How Non-Cytolytic Enteroviruses May Spread From Cell to Cell

[Hip]

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 lytic virus. 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, in the muscles in chronic inflammatory myopathy (an inflammatory muscle disease), and has been shown to infect the murine pancreas, which is relevant in udnertanding type 1 diabetes. 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.

Nora Chapman, Steven Tracy, John Chia, Patricia Tam and Ronald Messner and others pioneered investigation of chronic non-cytolytic enterovirus infections, and Chapman's research led to the discovery and understanding of the molecular mechanism behind non-cytolytic infection.



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 lytic enteroviruses, 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 (they produce viral proteins), and provoke an immune response, which may lead to disease.



How Non-Cytolytic Enteroviruses May Spread

This study demonstrates a possible mechanism by which non-cytolytic enteroviruses may be able to spread into adjacent cells.

This 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 CVB in order to transmit the infection into adjacent cells. In other words, enterovirus 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 CVB infection:

In the above video, cells infected with 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:

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.​

Some background info on the nature of cellular protrusions (click button):

Spoiler: Cellular Protrusions

Cellular protrusions are like the legs of the cell, as they allow a cell to move to specific locations in the tissues. Cellular protrusions comprise a portion of the normal membrane of the cell that has been elongated to form a long tendril shape.

There are two types of cellular protrusion:

Lamellipodia are broad and flat cellular protrusions which sprout out of the cell in the direction of intended 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.

Filopodia are long and thin cellular protrusions, and act as the guiding "eyes" of the cell, extending ahead of the cell and exploring the immediate environment of the cell, sensing guiding cues, and directing the movement of the cell.

Source: The mechanics of Cell Protrusion

More Info On Non-Cytolytic Enteroviruses

Non-cytolytic enterovirus — MEpedia.

Nora Chapman: How does a lytic enterovirus infection persist and cause chronic disease?

Human Enteroviruses and Chronic Infectious Disease by Prof Steven Tracy and Prof Nora M. Chapman.

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).

 

[globalpilot]

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

 

[Hip]

@globalpilot

Non-cytolytic enteroviruses do replicate slowly, but they have very little cytopathic effect — in other words they do not typically kill the cells they inhabit. But they can alter the functioning of the cells they infect, synthesize viral proteins, and the dsRNA from a non-cytolytic infection may elicit immune responses.

Interferon (IFN) can fight 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 it has been proposed (by Tam and Messner) that the non-cytolytic enterovirus dsRNA may make this infection very hard for the immune system to fight.

(Also 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).

Interferon alpha therapy for enterovirus-associated ME/CFS does often result in dramatic improvements in ME/CFS symptoms, but does not seem to fully eliminate non-cytolytic enterovirus infection from the tissues, so after a few months the infection grows back, and the patient then relapses.


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

In the above, TD viruses = terminally deleted coxsackievirus B = non-cytolytic coxsackievirus B

​

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

This 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 analogous to 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 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.

This positive stand RNA and negative strand RNA are then thought to be able to intertwine together to make double stranded RNA (dsRNA), which the immune system finds difficult to destroy. Dr Chia calls this dsRNA the "seeds", because dsRNA is tough and hardy in the face of an immune attack.

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. Ref: 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. Ref: 1



How Lytic Enteroviruses Turn Into Non-Cytolytic Enteroviruses

Non-cytolytic enteroviruses are created when certain specific mutations occur in the viral genome of lytic enterovirus during the acute infection stage. These mutations turn a virus that is normally only capable of acute infection into this non-cytolytic form which is able to create persistent intracellular infections of the tissues.

The transformation of the regular lytic enterovirus into its non-cytolytic form only occurs when enterovirus enters a non-dividing cell. This transformation does not take place when lytic enterovirus enters a dividing cell (like a liver cell).

Non-dividing cells (such as nerve, skeletal muscle or heart muscle cells), which are also called quiescent cells are key to the creation of a non-cytolytic enterovirus infection. The reason is that non-dividing cell provide an environment which favors the evolution and population growth of non-cytolytic enteroviruses over lytic enterovirus.

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



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. 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 difficult for the immune system to wipe out. It is proposed that the dsRNA can protect the non-cytolytic enterovirus infection from eradication during times of intense immune attack. When the immune response abates a little, it is likely the dsRNA can dissociate back to positive and negative ssRNA, and then recommence non-cytolytic viral replication.

• 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 about 100 times more positive than negative RNA strands).

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



Further Reading

More info on these non-cytolytic enteroviruses found in ME/CFS patients can be found in these video presentations and papers by Dr John Chia and Prof Nora Chapman:

Dr John Chia: The Role of Enterovirus in ME/CFS, Symposium on Viruses in CFS (video)

Dr John Chia: State of Knowledge Workshop on ME/CFS Research Part 1 (video)
Dr John Chia: State of Knowledge Workshop on ME CFS Research Part 2 (video)

Nora Chapman: How does a lytic enterovirus infection persist and cause chronic disease? (video)

Dr John Chia: The Role of Enterovirus in Chronic Fatigue Syndrome. 2005. J K S Chia


Google search for more info non-cytolytic enteroviruses.

 

[alex3619]

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.

 

[Hip]

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.

 

[Hip]

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.

I have long been interested in trying to understand the enterovirus infection that I think likely underlies the bulk of ME/CFS cases.

 

[cigana]

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?

 

[Hip]

Do you know if the antiviral used successfully by Lerner (Valtrex I think) is effective against the non-cytopathic forms?

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.

 

[frederic83]

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.

 

[Hip]

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.

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:

Parietal cells are terminally differentiated, polarized epithelial cells that are responsible for the acidification of the stomach.

Source: here

Numerous other studies have found non-cytolytic enterovirus in the skeletal muscles of ME/CFS patients.

 

[frederic83]

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.

 

[Hip]

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.

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.

 

[Gemini]

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.

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?

 

[frederic83]

They found nothing, because the samples were too old.

http://quixoticmeblog.blogspot.fr/2016/01/dr-c-on-hunt-again.html

 

[Wishful]

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.

 

[Hip]

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?

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, enterovirus RNA from non-cytolytic enterovirus was frequently found in the muscle tissues of ME/CFS patients (although at that time they did not know that non-cytolytic enteroviruses existed).

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.

 

[Gemini]

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?

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]

 

[Hip]

Has anyone seen a published paper on this work?

I have not seen any, but the CDC have a history of not really understanding what's going on when it comes to enterovirus testing in ME/CFS.

 

[perrier]

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]
Gemini
What would 'negative' mean??

 

[perrier]

I recall reading that Dr. Chia was in touch with Dr. Davis. Any news on that front??