Autopsy evidence of chronic EV infection Dr Chia Oct 2016

[Daffodil]

I'd say more annoying than scary, even if there is some silly virus with a stealth mechanism which is playing games with our brain cells, we would still need to find a way to explain how that could affect other areas of the body

well it would cause all kinds of inflammation, wouldn't it?

 

[Hip]

I recall way back in 1986 when I was shipped into a nursing home with severe ME Prof Mowbury (I don't think I've used the correct spelling) of London visited the home to take some of our thigh muscle fibres for the VP1 test to check for chronic enteroviral infection.

When I was diagnosed I was told ME was chronic coxsackie infection.

Amazing. Those were the days when the UK was a leading light in biomedical research into ME/CFS. Then quack self-promoting psychologists and psychiatrists hijacked the research and the country.

Many viruses have evolved countermeasures that allow them to evade the immune response.

@ash0787, the countermeasures that pathogens use to evade the immune response are known as immune evasion, in case you want to look this up.

it is scary realizing that we could have a virus in our brain tissue while it is not detectable anywhere else.

The viral infection is found elsewhere, particularly in the gut and the muscles. In this autopsy, they found an enterovirus infection in the stomach and colon tissues.


However, it is possible that the location of the infection may not be hugely important: if ME/CFS is largely caused by an autoimmune attack on our mitochondria, triggered by an enteroviral infection, then it would be the autoantibodies in the blood which are causing ME/CFS.

This is precisely what occurs in enterovirus infection of the heart (coxsackievirus B myocarditis) — in these infections, you get anti-mitochondrial autoantibodies that target heart cells, which leaves the heart cells low on energy. So in chronic coxsackievirus B myocarditis, the heart symptoms are not just a result of the ongoing infection in the heart muscle, but also a result of the autoantibodies which partially disable the mitochondria.

ME/CFS may be the same: some of the ME/CFS symptoms may come from an ongoing infection in the brain and other organs (with the inflammatory cytokines leading to symptoms); and other ME/CFS symptoms may come from anti-mitochondrial autoantibodies that whack our mitochondria.


It's possible that the anti-mitochondrial autoantibodies found in coxsackievirus B infection of the heart muscle are part of the immune evasion strategy of this virus: depleting cells of energy in this way may prevent the cells from clearing the virus (this study seems to suggest that).

 

[M Paine]

It has been found that enteroviruses can pass between cells without triggering cell lysis or even encapsidating into normal virion.

Do you have a source for regarding enteroviral transmission to cells that doesn't involve a virion?

 

[halcyon]

Do you have a source for regarding enteroviral transmission to cells that doesn't involve a virion?

There are several potential mechanisms.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246951/
https://www.ncbi.nlm.nih.gov/pubmed/24722773
https://www.ncbi.nlm.nih.gov/pubmed/26837894

 

[Hip]

Do you have a source for regarding enteroviral transmission to cells that doesn't involve a virion?

There is also the cellular protrusion route by which enterovirus RNA may spread from cell to cell (but this is hypothesized, but not proven at this point) — see these protrusions in the picture in this post.

Non-cytolytic enterovirus infections can also be seeded by normal lytic enterovirus infections, where a normal enterovirus virion enters a cell, but then under specific circumstances, it can change into a non-cytolytic infection (where the enteroviral RNA remains in the cell on a long term basis, and no virions containing lytic virus genomes are produced).

I believe you usually find lytic enterovirus infections coexisting in the same tissues as non-cytolytic infections; but in chronic infections, the non-cytolytic infections predominates over the lytic infection, which I think is why you can find RNA, but it can be hard to find any viral particles to culture.

 

[M Paine]

There are several potential mechanisms.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246951/
https://www.ncbi.nlm.nih.gov/pubmed/24722773
https://www.ncbi.nlm.nih.gov/pubmed/26837894

These are all papers detailing non-lytic pathways. They all still involve virions.

 

[M Paine]

My point is, that no VP1 RNA was able to be sequenced from any of the brain tissue samples. Any explanation involving infected cells selectively not expressing capsid protein, just doesn't make any sense. Even if the virus is not actively producing VP1 mRNA, it's still an +ssRNA virus. It would be there as part of it's genome, regardless of expression.

I think this investigation highlights why VirCapSeq-Vert is really going to be a valuable tool. I hope they get these samples to Dr Lipkin, and run them against more than the 2 selected primers.

 

[Hip]

My point is, that no VP1 RNA was able to be sequenced from any of the brain tissue samples.

I can't see where it says that in the autopsy text. But I may just be brain blind.


By the way, where is says in the text:

5’ EV RNA sequence was not detected by RT-PCR

This 5' (pronounced "five primed") region of the enteroviral genome is I believe the region that suffers some deletions when non-cytolytic RNA infections are generated. I don't understand it well, but non-cytolytic enterovirus genomes have some small deletions, and are not the full lytic virus genome.

I am not sure if that has any significance in non-detection of this 5' RNA sequence. Dr Chia knows a lot about non-cytolytic enterovirus, and he believes they play a major role in ME/CFS.


I have uploaded a pdf containing the slides of a presentation that Prof Nora Chapman gave on non-cytolytic enterovirus infection at the 2010 Invest in ME London Conference. This details these deletions a little. This presentation by Prof Chapman is also useful.

Or you can Google search on: 5' terminal deletions enterovirus. You will probably understand it better than I can.

 

[halcyon]

My point is, that no VP1 RNA was able to be sequenced from any of the brain tissue samples.

There is no VP1 RNA, the enterovirus genome produces one large polyprotein that is then cleaved into individual parts, such as VP1, VP2, the polymerases, etc.

There was actually VP1 protein expressed in the brain tissue samples though:

Using 5 D8/1 mAb, western blot revealed 37-42K and 46K protein bands in the brain samples, which corresponded to viral protein and creatine kinase b extracted from infected stomach biopsies, but not in brain biopsy samples taking from patients with brain tuberculoma and lymphoma.

5D8/1 is a monoclonal antibody that binds to all enterovirus VP1. It is known to cross react with brain type creatine kinase, but I believe when ran through a western blot they can be differentiated by their molecular weight.

One of the crucial points of this case study is that the initial attempt to isolate the virus with RT-PCR failed because the viral RNA was likely attached to DNA present in the sample. They had to apply DNase in order to free the RNA so that the PCR reaction could occur. This has implications that other researchers need to be aware of.

 

[M Paine]

Ah yes, excuse me. I had wrongly recalled that they sequencing for capsid proteins using VP1 primers. As you point out, they used 5' primers probably targeting the entire genome by targeting primers against the non-coding areas of the 5' end I guess. I haven't really looked into the genome of this Virus in detail. In any case, the point still stands. The virus still has these RNA sequences in it's entire genome, regardless of expressing VP1 precursors or not. It doesn't change the fact that they didn't get any hits, and really you have to ask the question, why not? The virus contains these sequences in it's genome. It happens to be a +ssRNA virus, which lends itself very nicely to these sorts of RNA probes. Regardless if the proteins are cleaved post translation, it doesn't matter at all. These primers can directly target the viral genome pre-translation, pre-cleavage.

 

[halcyon]

It doesn't change the fact that they didn't get any hits, and really you have to ask the question, why not?

They did get hits, just not on the initial PCR. I explained above briefly. The samples were ground and processed in medium. There is a known phenomenon where RNA can anneal to DNA. The initial RT-PCR was negative. They then added DNase which stripped the DNA off the RNA and tried again and were able to isolate the 3dpol part of the viral genome strongly in one sample. Additionally the samples were positive for VP1 protein by western blot so they knew that the virus almost certainly was there.

 

[M Paine]

Western blot alone doesn't confirm the presence of VP1. Regardless, I think it's more than fair to say that there are gaps in the evidence here when it comes to proving active infection of the brain of this person.

 

[halcyon]

Western blot alone doesn't confirm the presence of VP1.

How so? Isn't the whole point of western blot to demonstrate the presence or absence of a specific protein in a sample?

Regardless, I think it's more than fair to say that there are gaps in the evidence here when it comes to proving active infection of the brain of this person.

As I mentioned earlier, it doesn't necessarily need to be active to cause an immune response. The bare ssRNA or dsRNA alone in the cell will provoke an immune response via RIG-I and MDA5.

 

[M Paine]

So it's safe to say that the western blot confirms the presence of a VP1 like protein, or as least something of a comparable size which reacts to 5 D8/1 monoclonal antibodies.

What I was trying to get at above, which I phrased very poorly, was that the VP1 they are seeing, or whatever protein it is, is not necessarily the same virus observed in the intestinal tract of that patient. 5 D8/1 is not specific enough to make that determination.

If they had been able to sequence, or if there was a more specific mAb available, then sure. But it's entirely within the realm of possibility that the protein they saw was crossreactive and of similar size, or a VP1 protein from a virus different to Echovirus 11.

This is the kind of thing that requires validation, and more patient samples. Particularly brain tissue samples from non cfs controls who have had serious gastrointestinal infections of this sort. Brain tissue could be exposed to viral particles in those patients as well. We could potentially be seeing remnants of a non-permissive infection without deleterious effect.

 

[halcyon]

the VP1 they are seeing, or whatever protein it is, is not necessarily the same virus observed in the intestinal tract of that patient. 5 D8/1 is not specific enough to make that determination.

That's true, but it doesn't change anything if they match or don't match. It's possible to be superinfected with multiple serotypes at once, and they could easily be in different tissues.

If they had been able to sequence, or if there was a more specific mAb available, then sure. But it's entirely within the realm of possibility that the protein they saw was crossreactive and of similar size, or a VP1 protein from a virus different to Echovirus 11.

I assume because it's a poster presentation it's short on a bit of detail. This case was reviewed more in depth at IiME 2015. They did actually sequence the 3Dpol RNA that they isolated in the brain stem sample and it came back 86-92% homologous to coxsackie B2 and echovirus 30. I know nothing about enterovirus genome sequencing but it seems like it's never a dead on match due to how readily these viruses can mutate.

 

[RYO]

I spoke with researcher at University of Nebraska several years ago. He questioned the validity of VP1 staining of stomach and colon biopsies to determine whether someone has chronic EV infection.

I hope other research centers such as Stanford will take on the task of performing autopsy studies. It would be interesting if samples of vagus nerve were closely examined looking for viral genetic material.

I respect Dr. Chia's work but others in the scientific community need to repeat and verify his findings.

 

[halcyon]

I spoke with researcher at University of Nebraska several years ago. He questioned the validity of VP1 staining of stomach and colon biopsies to determine whether someone has chronic EV infection.

Did they say specifically why? The antibody used can cross react with several things, but I think we've moved far past that being a concern. It's been addressed by several researchers, including Chia. The tissue samples also can be shown to contain dsRNA and when lysates from the samples are injected into mice they transmit the infection, or straight up kill the mouse.

I respect Dr. Chia's work but others in the scientific community need to repeat and verify his findings.

Dr. Chia's work is itself a replication of Dr. Mowbray's research. I believe he was the first to create the 5D8/1 VP1 antibody and used it to demonstrate VP1 in the blood, GI tract, and brain samples of ME patients.

 

[halcyon]

It might be interesting to point out that almost 30 years ago, Dr. Mowbray showed that the molecular weight of echovirus 11 VP1 bound with 5D8/1 antibody is ~ 37 kDa, and they confirmed with radioimmunoprecipitation that it was indeed VP1 in their samples. This would seem to pretty closely match the weight of the protein in the western blot that Chia found here, and the patient was presumed to have echovirus 11 by serology.

 

[M Paine]

The antibody used can cross react with several things, but I think we've moved far past that being a concern

The presence of VP1 could be a range of Viruses. Most commonly, Poliovirus. People who received Oral Polio Vaccine (OPV) receive live attenuated vaccine, which replicates in the gut and is shed in stool, and can even spread to others in a community. The excreted PV which recipients excrete are more neurovirulent than the vaccine strain due to mutation and recombination in the gut.

In 2000, the US switched to IPV, but anyone vaccinated before that point, or people who came into contact with them, could easily have been exposed to live virus. In addition, many countries still use OPV.

 

[halcyon]

The presence of VP1 could be a range of Viruses. Most commonly, Poliovirus. People who received Oral Polio Vaccine (OPV) receive live attenuated vaccine, which replicates in the gut and is shed in stool, and can even spread to others in a community. The excreted PV which recipients excrete are more neurovirulent than the vaccine strain due to mutation and recombination in the gut.

Anything is possible. My understanding though is that OPV reversion is quite rare. You'd still have to explain why there is a significantly greater amount of ME patients with VP1 in their stomach than healthy controls, per Chia's research.

Regardless, in this case the virus from the brain sample was shown to most likely be HEV-B not HEV-C. In fact I'm not aware of any ME research where the enterovirus isolated was anything other than one resembling HEV-B serotypes, other than one study that actually found novel 5' enterovirus sequences in the ME patients.