XMRV and Culturing, HERV's and more

[jackie]

Dear folks...since I'm seeing a collection of really brilliant minds - all here, all in one place - debating XMRV...I don't suppose we might segue into a discussion of Dr. John Chia's Enteroviruses and THOSE possibilities - re:XMRV? (That's with YOU discussing...and ME listening attentively! - I am just the guinea pig).

OR if I started a new thread, would any here care to do some speculating?

Dr. Chia believes that Enteroviruses are involved in me/cfs, and he ultimately identifies Enteroviral presence through stomach biopsies - which proves that these viruses continue to live in the gut ecology (DIS-proving that Enteroviruses are "hit and run viruses"!). He finds Enteroviral involvement in 82% of his CFS patients - and 20% of his controls.)

He also discovered an antibody test for Coxsackie and Echoviruses that is more specific than other labs. (By "serendipity" a sample from his offce ended up in ARUP Lab in Salt Lake City - and the results came back quite positive. Upon investigation, he discovered that this ARUP Lab does a test that is more specific than other labs....reporting antibody levels of Coxsackie B1-6 and 5 of the 27 Echoviruses. An elevation of 1:320 is considered to be significant - for B-4, especially. If one is unable to get a stomach biopsy, then I THINK a key is to get the antibodies test.)

So far, Dr. Chia has been able to PROVE the existance of Enteroviruses in the gut of his patients. (Coming as a "surprise" to some in the me/cfs community...as many have no clue about this!)
BTW...this is ONE of the docs/researchers that KNOWS me/cfs...and truly believes "us"!....and has dedicated his LIFE to finding the cause, treatment and hopefully a cure one day...he just works quietly - on and on!

He speculates that if Enteroviruses are in the GUT - then they could also be in the HEART, the THYROID, the PANCREAS - AND the BRAIN! Proving the existance of Enteroviruses in TISSUE is a big deal (but it does NOT establish that it is the CAUSE of me/cfs - yet). However, Dr. Chia assumes that enteroviruses are a cause in a LARGE proportion of cases.

(Although his ideas are specific and scientifically proven - are we putting aside some serious research that is "staring us right in the face"....by discounting his "re-forged" connection between ME/CFS and Enteroviruses?)

The unfortunate part of this situation is that WITH this clear IDENTIFICATION of a POTENTIAL cause of me/cfs - there is NO funding available to develop anti-ENTEROVIRAL drugs! (he says at this rate, it will be 10 years before such drugs "will be forthcoming").
Might it be worthwhile to take a step BACKWARDS and look (once again) at the possibilty that Enteroviruses are heavily involved in our disease, WITH or WITHOUT XMRV?.

Ideas, anyone? Interest, anyone?

thanks, j

(much of this "info" came from interviews found here on the Forums, the CFS Patient Advocate Blog, and the EV Foundation - paraphrased by ME!)

 

[Gerwyn]

Gerwyn please respond to the fact that WPI researchers apparently did not culture their samples before PCR testing yet now Dr. Mikovits states that culturing is absolutely necessary. This is how this got started - as a way to explain this oddity. Do you agree that there is no evidence in the paper that the PCR samples were cultured first?

If not then please give us some idea of why culturing would be necessary later on?

I would note that this could be the reason Dr. Vernon came down so hard on the WPI for not telling us more about those original patients. She noted that first they didn't need to culture and then they did - that could suggest that those patients were different; that they were much sicker and thus the virus was much easier to find. That is one interpretation is it not?

The virus was only detected by the PCR method used because the pmbc cells were concentrated and activated to induce replication. At this stage the presence of live viable virus could not be confirmed It could have been a herve for example.The culturing and transfection step was neccessary in this context to prove the existence of a live replicative exogenous virus.

As for the need for culture now the situation is identical to the situation in the diagnosing the presence of HIV PCR alone leads to an unacceptably high number of false negatives

The culturing process is carried out for different reasons in the two instances

I am at a loss to explain Dr Vernons attitude to the WPI trial.

There is much more ambiguity re patient characteristics in the European studies . This could easily explain the differences in results,without the obvious differences in methodology.

I am even more suprised given that Dr vernon is a virologist .

The WPI method is clearly outlined albeit with some paragidm specific terms which any currently practising virologist would understand anyway.

I understand that Dr vernon has been in a managerial position and not actively involved in virology for some years.

Some of the specific terminology used by retrovirologists may be giving her some difficulties.

I have not practised microbiology for some years and the terms confused me at first until I researched theirspecific meaning

 

[Gerwyn]

what kind of enteroviruses has he found thus far

 

[Esther12]

There is much more ambiguity re patient characteristics in the European studies . This could easily explain the differences in results,without the obvious differences in methodology.

Thanks for your comments on the culturing (although it's a bit over my head), but I don't think patient selection could easily explain the differences in results. Unless the WPI had restricted itself to the housebound, or sickest of the sick, the first couple of studies would have been really unlikely to include no patients matching the WPI's criteria for CFS. The failure to detect XMRV in these studies surely stems from either different methods being used, or a problem with the WPI's results.

 

[Cort]

The virus was only detected by the PCR method used because the pmbc cells were concentrated and activated to induce replication. At this stage the presence of live viable virus could not be confirmed It could have been a herve for example.The culturing and transfection step was neccessary in this context to prove the existence of a live replicative exogenous virus.

Thanks I appreciate that. Now the question is if the other studies concentrated and activated the PBMC's to induce replication? Do you know if they did that?

There's also the question why culturing appears to be deemed necessary now but wasn't before. What could account for this? Lower levels of the virus in the new patients? From your reply I can see that concentrating and activating PBMC's is necessary but I don't see why they wouldn't continue doing this. I assume something else must be a factor?

 

[ukxmrv]

Jackie,

Could you start another thread on retroviruses?

The theory that they cause ME have never died in the UK and is still alive and well among patients and old ME doctors. We even had a VP1 blood test for it on our NHS system in the late 80's

Would be best to keep the discussion together on one thread as this is XMRV

 

[Gerwyn]

Thanks I appreciate that. Now the question is if the other studies concentrated and activated the PBMC's to induce replication? Do you know if they did that?

There's also the question why culturing appears to be deemed necessary now but wasn't before. What could account for this? Lower levels of the virus in the new patients? From your reply I can see that concentrating and activating PBMC's is necessary but I don't see why they wouldn't continue doing this. I assume something else must be a factor?

no that is the point they did not

culture is a simpler cheaper way of amplification which can be done in all commercial labs.in the WPI the other aplification proceedures enabled characterisation of the virus by various methods DNA typing etc.Also at the time they did not know that the virus was a factor at all.Now that various parameters have been investigated there is no need to use the more specialised proceedures to amplify.

 

[jackie]

Gerwyn! I was hoping that you would see this (if not have not already investigated).
I don't even know how to give you links...I would be glad to - if someone can tell me how!

Dr. Chia has a website with a research page...."EVMED research". There he mentions the different ent's and the difficulties in finding them and the methods used. I copied the "CONCLUSION" to ONE his many studies (as I realise you need facts).."Enterovirus VP1 (Viral Capsid Protein 1), RNA and Non-cytopathic Viruses were detected in the stomach specimens of CFS patients with Chronic Abdominal complaints. A significant subset of cfs patients may have a chronic, disseminated, non-cytolytic form of enteroviral infection."

Please let me know if you want me to get the info for you or (if you are interested) you'd rather look it up yourself on his evmed site. Very exciting stuff, imo.

I'm one of his patients on long term, high dose Acyclovir (4 yrs./3200mgs. daily). Interesting side note re Dr. Chia's involvement with enteroviruses (he formerly worked exclusively on EBV for many years) -he does not claim originality in his study of enteroviruses...instead he builds on a forgotten but established legacy of research in the UK, partucularly of John Richardson (d.2003). Coxsackie and other enteroviruses were on the frontlines of me/cfs research until they faded away from the picture for a variety of reasons...he is attempting to bring them back into the "picture"!

As for me, one of the areas of NOTICEABLE (to ME, anyway...if not to anyone else!) improvement is with Memory/cognitive. AND, PEM does NOT last as long (and is NOT as severe).
I continue to have many other problems (chronic reactivated shingles on BOTH sides...BUT they are quite manageable now!, hsv1, high EBV, CMV titres, etc.). I was instructed the other day to begin equilibrant NOW (newer version of Oxymatrine), in addition to Antiviral...to discontinue (for the time being) Tagamet.

The HHV6 Foundation also has a patient forum with some discussion of Oxymatrine use.
thanks for your interest! jackie

 

[jackie]

OOPS! sorry, ukxmrv...just saw YOUR post! I guess this IS in the wrong place...hope I didn't hi-jack anybody! (the only reason I originally posted in the OTHER thread (before it was "split"...i'm sort of confused!)...it seemed as though OTHER possibilities were being raised...and I wondered about adding Enteroviruses to the XMRV mix! Should I start a thread on "ENTEROVIRUSES"? of "RETROVIRUSES"? (i need a nap! j)

 

[Gerwyn]

Gerwyn! I was hoping that you would see this (if not have not already investigated).
I don't even know how to give you links...I would be glad to - if someone can tell me how!

Dr. Chia has a website with a research page...."EVMED research". There he mentions the different ent's and the difficulties in finding them and the methods used. I copied the "CONCLUSION" to ONE his many studies (as I realise you need facts).."Enterovirus VP1 (Viral Capsid Protein 1), RNA and Non-cytopathic Viruses were detected in the stomach specimens of CFS patients with Chronic Abdominal complaints. A significant subset of cfs patients may have a chronic, disseminated, non-cytolytic form of enteroviral infection."

Please let me know if you want me to get the info for you or (if you are interested) you'd rather look it up yourself on his evmed site. Very exciting stuff, imo.

I'm one of his patients on long term, high dose Acyclovir (4 yrs./3200mgs. daily). Interesting side note re Dr. Chia's involvement with enteroviruses (he formerly worked exclusively on EBV for many years) -he does not claim originality in his study of enteroviruses...instead he builds on a forgotten but established legacy of research in the UK, partucularly of John Richardson (d.2003). Coxsackie and other enteroviruses were on the frontlines of me/cfs research until they faded away from the picture for a variety of reasons...he is attempting to bring them back into the "picture"!

As for me, one of the areas of NOTICEABLE (to ME, anyway...if not to anyone else!) improvement is with Memory/cognitive. AND, PEM does NOT last as long (and is NOT as severe).
I continue to have many other problems (chronic reactivated shingles on BOTH sides...BUT they are quite manageable now!, hsv1, high EBV, CMV titres, etc.). I was instructed the other day to begin equilibrant NOW (newer version of Oxymatrine), in addition to Antiviral...to discontinue (for the time being) Tagamet.

The HHV6 Foundation also has a patient forum with some discussion of Oxymatrine use.
thanks for your interest! jackie

yes please if you can send info it would be great another class of RNA viruses almost impossible to detect by conventional assay methods

 

[Gerwyn]

Thanks for your comments on the culturing (although it's a bit over my head), but I don't think patient selection could easily explain the differences in results. Unless the WPI had restricted itself to the housebound, or sickest of the sick, the first couple of studies would have been really unlikely to include no patients matching the WPI's criteria for CFS. The failure to detect XMRV in these studies surely stems from either different methods being used, or a problem with the WPI's results.

The dutch study did not have any cfs patients in it.Patients diagnosed according to the oxford criterea can easily have fatigue caused entirely caused by depression.One of the authors of the criterea has gone on public record as saying so.

 

[Esther12]

The dutch study did not have any cfs patients in it.Patients diagnosed according to the oxford criterea can easily have fatigue caused entirely caused by depression.One of the authors of the criterea has gone on public record as saying so.

Sorry - by first couple of studies I meant the Wessely and Kerr ones. The dutch study's patient selection could have been a problem.

 

[natasa778]

Enteroviruses in ME/CFS, Diagnosis and Treatment

On the subject of Dr John Chia, he will be presenting on this in May in London. Judy will also be there so should make for an interesting Q&A session, if they have time for one.

http://www.investinme.org/IIME Conference 2010/IIME 2010 International ME Conference Agenda.htm

 

[jackie]

Yes, Natasha! This next conference should be VERY interesting! (my next appointment with Dr. Chia is right before he leaves!) Gerwyn...I'm collecting "stuff" for you! (note: a major drawback of the serological testing is that neutralizing antibody tests are only available for 11 of the 70+ non-polio enteroviruses.)

 

[Hope123]

My personal take on all this is: time will tell. We can debate all we want but in the face of no recent new studies, it's all conjecture. And I also agree that unconfirmed information can be harmful, either by getting people's hopes up too much or dashing them without any real evidence. Thus, in my past work life and now, if I don't get a direct statement that something is public, I do not make it public.

Re: EM studies. This can be resolved somewhat by staining the suspected retroviral particles seen with antibodies to either XMRV or HERVs to try to characterize what they are.

And I agree with posters who say that even if these are HERVs, the question then becomes what is activating them? The classic example of superantigens is toxic shock syndrome which results from a superantigen toxin put out by the bacterium staphylococcus aureus. People die from it because of the cytokine storm created. The treatment is quick diagnosis and antibiotics aimed at S. aureus. IIRC, this was the first superantigen found.

The other possible way to suss out these issues is to consider tissue staining for XMRV in tissues of people with CFS much like the primate experiments.

 

[natasa778]

The other possible way to suss out these issues is to consider tissue staining for XMRV in tissues of people with CFS much like the primate experiments.

This is hugely important imo, I've been saying all along we won't have real and usable (as in pathology etc) answers until many tissue biopsies and postmortem studies are carried out. Gut lining for both enteroviruses and retroviruses, brain vascular endothelium, inc microglia astrocytes where cognitive issues present ...

 

[Cort]

I see this is in Imperial College:

DNA was extracted from EDTA whole blood using a standard phenol-based organic deproteinisation procedure [19]. DNA concentrations were determined by absorbance at 260 nm (A260). Each sample was amplified in three nested PCRs using primers targeted to an XMRV-specific sequence, to a sequence conserved amongst most MLV and, as a control for sample addition and PCR-inhibition, to a human beta-globin (hBG) sequence (Table 1).

I see they used whole blood instead of the targeting lymphocytes. Then did they amplify differently?

Here's Groom

Genomic (g)DNA was prepared from PBMC from SGUL patients and controls using the QIAamp DNA mini kit (Qiagen) and amplified using the RepliG Ultrafast Mini Kit (Qiagen), which provides highly uniform amplification of all sequences, with negligible sequence bias. The concentrations after amplification ranged from 108 - 586 ng/μl. Initially, 48 CFS patient gDNA samples were screened by single-round PCR for gag and env genes, as well as GAPDH, as outlined by Lombardi et al. [8]​

Kuppenfeld

Blood samples were sent to the central laboratory of the blood transfusion service in Amsterdam, where peripheral blood mononuclear cells were isolated for a study of lymphocyte subsets and apoptosis.1​

There's Was the most complex but I don't see anything on amplification.

 

[Gerwyn]

I see this is in Imperial College:

I see they used whole blood instead of the targeting lymphocytes. Then did they amplify differently?

Here's Groom

they did not activate the rest is history no activation no cigar

 

[Gerwyn]

I see this is in Imperial College:

I see they used whole blood instead of the targeting lymphocytes. Then did they amplify differently?

Here's Groom

sorry cort i missed it amplyfy has a different meaning here this is PCR not sample amplification can be confusing I know

 

[Cort]

It is so complex. I appreciate your insights.

We'll see in the end what is going on, I believe. There should be quite a few more studies - each with different procedures I imagine - and hopefully better patients sets. I actually heard that several papers are in press.

Here's from the Science Paper

PCR amplification for sequencing full-length XMRV genomes was performed on DNA amplified by nested or semi-nested PCR from overlapping regions from PBMC DNA. For 5’ end amplification of R-U5 region, 4F (5’CCAGTCATCCGATAGACTGAGTCGC-3’) and 1154R was used for first round and 4F and 770R (5’-TACCATCCTGAGGCCATCCTACATTG-3’) was used for second round

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I guess the activation of the lymphocytes - to get them to start producing viruses - comes earlier. Here's what they did:

The PBMC (approximately 2 x 10 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis.

They were either stored as unactivated cells for the later culturing process or stored as is for DNA/RNA extraction. It doesn't look like any activation for the PCR ?