XMRV and Culturing, HERV's and more

[Gerwyn]

It is so complex. I appreciate your insights. So the WPI used amplification/activation - need to activate those cells - before and is using culturing now and they're basically the same thing and they argue that everyone needs to use one or the other - and they're not - is that your take on this? And none of the things I showed up are the type of amplification required?

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

both amplification(of sample) and activation(ofpmbc) are needed.The concentration of virus needs to be increased and coaxed into replication .Yes amplification of virus concentration is different from PCR amplification cycles.

If any trial does not include these steps then the chances of finding the bug will be minimal.The WPI method itself would not find XMRV in patients diagnosed according to the Oxford criterea because it is not thought to be associated with people with purely psychological problems

 

[dannybex]

The WPI method itself would not find XMRV in patients diagnosed according to the Oxford criterea because it is not thought to be associated with people with purely psychological problems

With all due respect, this is not true. The WPI found 3.7 percent of 'healthy' controls to have XMRV...which equals approximately 10 million people without CFS/ME in the US alone.

???

 

[Dr. Yes]

It is so complex. I appreciate your insights. So the WPI used amplification/activation - need to activate those cells - before and is using culturing now and they're basically the same thing and they argue that everyone needs to use one or the other - and they're not - is that your take on this? And none of the things I showed up are the type of amplification required?

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

Hi Cort,

I believe what Gerwyn originally meant by amplification was what is referred to in the Science paper as activation; he was originally using the term amplification in a broad sense for any attempt to increase any target (be it whole cells or DNA sequences). For the purposes of these XMRV papers, it may be better to use the term amplification the way they did - to refer only to PCR amplification. All PCR techniques involve amplification of a given DNA sequence; that's the basis of PCR. One thing the WPI did differently was to activate primary cells, i.e. stimulate them to replicate quickly; for this they cultured them with IL-2 (interleukin-2). IL-2 is a cytokine which in our bodies is used as a signal to stimulate the immune response.

What you mentioned were PCR amplification procedures, but as I noted all PCR techniques use them. The key here is how to increase cell numbers (and therefore viral copy number) to a level that allows for viral detection; this is done by activation of cells taken from patients, i.e getting them to replicate. That is the step missing, as far as I can tell, from the UK and Dutch studies.

Hope that helps clear things up. It would be impressive if it did; my brain is totally fried.

 

[Gerwyn]

With all due respect, this is not true. The WPI found 3.7 percent of 'healthy' controls to have XMRV...which equals approximately 10 million people without CFS/ME in the US alone.

???

the point is that oxford selection criterea can easily select patients who dont have cfs at all but have fatigue caused by psychological factors as per the dutch study for example

 

[ukxmrv]

It doesn't really matter if Mikovits et al missed out reporting the step of the culture of the PCR cells using Lncap in the original Science paper. She can answer that question and the record can be put straight.

It is important for groups planning replications but they could have got the information from her (I've had my own correspondence and am aware of her very helpful attitude toward one researcher). She's been answering my own basic questions and I've been copied into other correspondence so it's not "hearsay" for me.

It's also important to have as much information in the public domain but this can be covered by questions. Science is going to do this. It appears to be that some people are nit-picking and deliberately making mountains out of molehills.

The paper doesn't need to be retracted. That's an over reaction. It's just a clarification and everyone wants to see the method explained well.

In our own UK testing we are seeing many XMRV+ positive people. There is nothing unusual about these patients. Just a group of people with CFS and ME who wanted to be tested and could afford to do so.

I'm XMRV+ and from a known outbreak of ME, that occurred before CFS was named. I was PCR-, culture +. That doesn't bother me having seen the Japanese abstract and have corresponded with Dr Mikovits.

Yes, it is important that we determine the XMRV, HERV question. No one, including me wants to go down a blind alley and waste valuable time.

None of the concerns here invalidate the paper. There are questions to be answered and things may turn out to be different than we think right now.

Let's see how the Science questions go. I'm not asking any further of Dr M as I know she is busy on the UK study. I have a feeling that some people (not necessarily here) will never be happy with the replies and the nitpicking will continue regardless.

 

[Gerwyn]

Yes I mean that the act of activation amplyfied the number of PMBC and thus viral nucleic acid load and ensured that the viruses became replicative producing the cDNA needed if PCR was to work Sorry I can go into shorthand as well

 

[Cort]

That was actually very clear Dr. Yes ( as your posts usually are ) - it did clear some things up for me. I think your brain is doing pretty darn good. But I don't see any evidence of 'activation' as pertains to the PCR section of the paper.

It could be that I'm missing something. If I'm not then could it be that activation was not required in the early patients (because their viral loads were higher?) but is required in the patients they're seeing now? As I mentioned earlier if this is true then it would provide a good reason why Dr. Vernon has gotten so fixated on that original cohort?

Because I know everybody loves reading this stuff - here's the entire methods section leading up to the PCR test

DNA and RNA isolation. Whole blood was drawn from subjects by venipuncture using standardized phlebotomy procedures into 8-mL greencapped Vacutainers containing the anti-coagulant sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Plasma was collected by centrifugation, aspirated and stored at -80 C for later use. The plasma was replaced with PBS and the blood resuspended and further diluted with an equal volume of PBS. PBMC were isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and washing it once in PBS. 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.DNA was isolated from TRIzol preps according the to manufacturer’s protocol and also isolated from frozen PBMC pellets using the QIAamp DNA Mini purification kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol, and the final DNA was resuspended in RNase/DNasefree water and quantified using the Quant-iT Pico Green dsDNA Kit (Invitrogen, Carlsbad, CA). RNA was isolated from TRIzol preps according to the manufacturer’s protocol and quantified using the Quant-iT Ribo Green RNA kit (Invitrogen, Carlsbad, CA). cDNA was made from RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol.

PCR. To avoid potential problems with laboratory DNA contamination, nested PCR was performed with separate reagents in a separate laboratory room designated to be free of high copy amplicon or plasmid DNA. Negative controls in the absence of added DNA were included in every experiment. Identification



3
of XMRV gag and env genes was performed by PCR in separate reactions. Reactions were performed as follows: 100 to 250 ng DNA, 2 L of 25 mM MgCl2, 25 L of HotStart-IT FideliTaq Master Mix (USB Corporation, Cleveland, OH), 0.75 L of each of 20 M forward and reverse oligonucleotide primers in reaction volumes of 50 L. For identification of gag, 419F (5’ATCAGTTAACCTACCCGAGTCGGAC-3’) and 1154R (5’GCCGCCTCTTCTTCATTGTTCTC-3’) were used as forward and reverse primers. For env, 5922F (5’- GCTAATGCTACCTCCCTCCTGG-3’) and 6273R (5’-GGAGCCCACTGAGGAATCAAAACAGG-3’) were used. For both gag and env PCR, 94C for 4 min initial denaturation was performed for every reaction followed by 94C for 30 seconds, 57C for 30 seconds and 72C for 1 minute. The cycle was repeated 45 times followed by final extension at 72C for 2 minutes. Six microliters of each reaction product was loaded onto 2% agarose gels in TBE buffer with 1 kb+ DNA ladder (Invitrogen, Carlsbad, CA) as markers. PCR products were purified using Wizard SV Gel and PCR Clean-Up kit (Promega, Madison, WI) and sequenced. 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. For regions including gag-pro and partial pol, 350F(5’GAGTTCGTATTCCCGGCCGCAGC-3’) and 5135R (5’- CCTGCGGCATTCCAAATCTCG-3’) was used for first round followed by second round with 419F and 4789R (5’-GGGTGAGTCTGTGTAGGGAGTCTAA-3’). For regions including partial pol and env region, 4166F (5’- CAAGAAGGACAACGGAGAGCTGGAG-3’) and 7622R (5’GGCCTGCACTACCGAAAT TCTGTC-3’) were used for first round followed by 4672F (5’GAGCCACCTACAATCAGACAAAAGGAT-3’) and 7590R (5’- CTGGACCAAGCGGTTGAGAATACAG-3’) for second round. For the 3’ end including the U3-R region, 7472F (5’-TCAGGACAAGGGTGGTTTGAG-3’) and 8182R (5’-CAAACAGCAAAAGGCTTTATTGG-3’) were used for first round followed by 7472F and 8147R (5’-CCGGGCGACTCAGTCTATC-3’) for second round. The reaction mixtures and conditions were as described above except for the following: For larger fragments, the final extension was done at 68C for 10 min instead of 72C for 2 min. All second round PCR products were column purified as described above and overlapping sequences were determined with internal primers. oNested RT-PCR for gag sequences was done as described (5) with modifications. GAG-O-R primer was used for 1st strand synthesis; cycle conditions were 52oC annealing, for 35 cycles. For second round PCR, annealing was at 54C for 35 cycles. PCR analysis performed on 20 of the identical patient PBMC DNA specimens stored at the NCI (Frederick, MD) since 2007confirmed nearly identical gag sequences, thereby diminishing the possibility of laboratory contamination as a source of XMRV.

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[dannybex]

One thing the WPI did differently was to activate primary cells, i.e. stimulate them to replicate quickly; for this they cultured them with IL-2 (interleukin-2). IL-2 is a cytokine which in our bodies is used as a signal to stimulate the immune response.

Very interesting Dr. Yes. My brain is totally fried too, but isn't IL-2 one of the cytokines that has been found to be very low in PWC's? (Or PWME's?) Perhaps XMRV is something that lowers these cytokine levels...I don't know.

Here's a study that found undetectable IL-2 levels in the spinal fluid of PWC's:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC540195/

d.

p.s. thanks Gerwyn. I knew that was what you meant...makes sense...but one would think if WPI had done the testing, they would find at least some with XMRV.

 

[dannybex]

because i know everybody loves reading this stuff :retro rolleyes: - here's the entire methods section leading up to the pcr test

"20 m forward and reverse oligonucleotide primers in reaction volumes of 50 l. For identification of gag, 419f (5atcagttaacctacccgagtcggac-3) and 1154r (5gccgcctcttcttcattgttctc-3) were used as forward and reverse primers. For env, 5922f (5- gctaatgctacctccctcctgg-3) and 6273r (5-ggagcccactgaggaatcaaaacagg-3) were...nested or semi-nested pcr from overlapping regions from pbmc dna. For 5 end amplification of r-u5 region, 4f (5ccagtcatccgatagactgagtcgc-3) and 1154r was used for first round and 4f and 770r (5-taccatcctgaggccatcctacattg-3) was used for second round. For regions including gag-pro and partial pol, 350f(5gagttcgtattcccggccgcagc-3) and 5135r (5- cctgcggcattccaaatctcg-3) was used for first round followed by second round with 419f and 4789r (5-gggtgagtctgtgtagggagtctaa-3). For regions including partial pol and env region, 4166f (5- caagaaggacaacggagagctggag-3) and 7622r (5ggcctgcactaccgaaat tctgtc-3) were used for first round followed by 4672f (5gagccacctacaatcagacaaaaggat-3) and 7590r (5- ctggaccaagcggttgagaatacag-3) for second round. For the 3 end including the u3-r region, 7472f (5-tcaggacaagggtggtttgag-3) and 8182r (5-caaacagcaaaaggctttattgg-3) were used for first round followed by 7472f and 8147r (5-ccgggcgactcagtctatc-3) for second round....."

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MY EYES...MY EYES!!!..

:Retro smile:

 

[kurt]

I have been told the importance of amplification varies with the sensitivity of the test. For a less sensitive PCR more amplification prior to testing may be critical, as Gerwyn says, getting the cells to replicate the virus. However, some of the other studies after WPI used more sensitive real-time PCR tests, I believe one of the UK studies mentioned their test could detect a single virus. Therefore amplification might be less critical, as long as DNA was properly extracted.

No doubt more of the pending studies will be using more sensitive tests than the WPI version, which was a standard PCR. Certainly the researchers involved all understand the concentration/sensitivity issue, I don't think that is the only problem here.

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.

Yes, time will tell. Meanwhile the WPI hypothesis is also still unconfirmed information. Just pointing that out, the caution about getting people's hopes up or dashing them goes both ways.

If you want to know about the HERV K18 superantigen in CFS, you might read some of Dr Bridgette Huber's work. That can be activated apparently by HHV, and I have read that HERVs are also more like to activate when methylation is failing (a methylation stage is involved in keeping HERVs quiet).

What you mentioned were PCR amplification procedures, but as I noted all PCR techniques use them. The key here is how to increase cell numbers (and therefore viral copy number) to a level that allows for viral detection; this is done by activation of cells taken from patients, i.e getting them to replicate. That is the step missing, as far as I can tell, from the UK and Dutch studies.
Hope that helps clear things up. It would be impressive it did; my brain is totally fried.

This is what I was trying to say above. The European study authors probably thought their tests were sensitive enough, at the level that allows for viral detection. They had the count levels and sensitivity and sample volume information from Science. Figuring if their test might work was basic math, and they obviously thought the tests should have worked. And they DID work on positive controls. But they got no PCR hits, so AT THAT RESOLUTION nothing was detected.

It doesn't really matter if Mikovits et al missed out reporting the step of the culture of the PCR cells using Lncap in the original Science paper. She can answer that question and the record can be put straight.

Maybe it does not matter to you but I think that would matter a lot to the many labs who have spent hundreds of thousands of dollars of their budgets running tests based on the Science paper. What is published peer review like this must be adequate information. These labs, particularly private ones but also University, are all competitors. We do not know their real relationships of these labs behind the scenes. Anyway, finding a specified virus in a specified population is a very generic finding, labs should be able to validate that on their own given the information in a journal article. If Mikovitzs has to give 'extra' information behind the scenes, then the Lombardi et al article was incomplete.

 

[kurt]

Keep in mind that the WPI Science paper very clearly stated that the patients were severely affected, Canadian and Fukuda defined patients with exercise and immune abnormalities (NK, Rnase L, cytokine and VO2 stress test results). All of the early researchers completely ignored this important information. So far hundreds of thousands of dollars have been wasted on unusual 'CFS' cohorts that do not resemble the original cohort.
Hopefully we will get some Canadian defined patients in studies before the end of the year. At that point we should have a clearer idea of what is going on.

Yes of course, any cohort definition part of their failure was the fault of the labs. I was only speaking to the possibility of techniques that may have been missing from the article. That would be on WPI and is a serious mistake. At this point we do not have enough facts to draw well-supported conclusions in either direction.

 

[julius]

Hey Kurt. I am wondering how the HERV theory squares with the statement by Dr Mikovits that they sequenced the entire XMRV genome two and a half times. It seems to me that with the whole genome you would know for certain if you were looking at a HERV or XMRV.

 

[Gerwyn]

Hey Kurt. I am wondering how the HERV theory squares with the statement by Dr Mikovits that they sequenced the entire XMRV genome two and a half times. It seems to me that with the whole genome you would know for certain if you were looking at a HERV or XMRV.

yes you would I have looked at the sequencing a herve would stand out like a sore thumb

 

[Gerwyn]

Hey Kurt. I am wondering how the HERV theory squares with the statement by Dr Mikovits that they sequenced the entire XMRV genome two and a half times. It seems to me that with the whole genome you would know for certain if you were looking at a HERV or XMRV.

yes you would I have looked at the sequencing a herv would stand out like a sore thumb

 

[Gerwyn]

This following comes from the Imperial college study

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician .Detectable by blood tests OR patient self reporting This is what taking a patients history means.

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician . So Detectable by blood tests OR patient self reporting ..This means that the patients had none of the symptoms reported by the patients in The WPI cohort OR any of the biochemical or immunological annormalities. By McCure,s own introduction they are an entirely different cohort to those investigated by the WPI


DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR

Now this seems an innocuous statement does it not?

The point to realise is that she knew about Mulv and the fact that it was closely related to XMRV.

As a virologist surely she would know that Mulv does not replicate when in the bloodstream.It only replicates in lymphoid tissue,after replication it quickly becomes latent..

So knowing this she attempts to isolate the XMRV DNA directly from the blood by PCR even though she knows that this would not be possible with Mulv

She knows that Mulv lives in lymphoid cells but chose not to examine lymphoid cells for XMRV content at all

To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

Ok the concentration of Beta globulin gene is many orders of magnitude higher than the concentration of cDNA would be This proves nothing at all



Received: December 1, 2009; Accepted: December 4, 2009; Published: January 6,

2010

Peer review in 4 days could be as little as one .They invite you to believe that underwent peer review in the period between the two dates.How can anyone anyone review a paper in this time.The Science peer review took months

 

[Gerwyn]

Hi Esther,

Have you read the two CBT studies from which Wessely drew his 186 samples? I think it's important to review these two studies before arguing that Wessely's patients were in any way comparable to the WPI Science cohort. Unfortunately the two papers are not freely available but if you can get access, these are the details:

can anyone get me these papers please

 

[Knackered]

Hopefully we will get some Canadian defined patients in studies before the end of the year. At that point we should have a clearer idea of what is going on.

Before August I'd have thought.

 

[Gerwyn]

This embarrassing question is addressed as part of the anti-XMRV season at the BMJ. Richard Smith, former BMJ chief editor and now a member of the PLOS board, wants to scrap peer review because, "Much of what is published in journals is scientifically pooras the Science article shows."

i thought that Plos one had already scrapped the peer review process

 

[Gerwyn]

Yes of course, any cohort definition part of their failure was the fault of the labs. I was only speaking to the possibility of techniques that may have been missing from the article. That would be on WPI and is a serious mistake. At this point we do not have enough facts to draw well-supported conclusions in either direction.

We have more than enough information to draw conclusions about the cohort issues

 

[Gerwyn]

(This thread picked up from the "Dr. Vernon thread on the General News Forum": http://www.forums.aboutmecfs.org/sh...trists-from-the-Wesselly-school-as-Co-Authors)



So you have no beliefs here? No speculation? I would say you have shown plenty of belief in your posts. I only say I believe something when I do not have the references handy to quote, but most of what I say I can back up with data. Just don't have the time to sort through it all continually, unless I am writing a paper. I could write out all the facts I am working from, probably have mentioned most of them already. But regarding current issues, the most important fact is that I know many people with CFS who are testing negative for XMRV, and they DO have CFS but do not have XMRV, including by WPI, and other outside labs that hopefully will eventually publish their studies.

Yes, a systems model can really help in a complex situation like this. Systems biology is the study of interactions between parts of a biological pathology. Think of the 'sum of the parts being greater than the whole' but apply that to a disease model. I believe CFS is a perfect match-up with the systems biology paradigm. I believe there is a wiki entry on systems biology.

[Gerwyn, an aside, you continue to use put-downs, and a condescending attitude in your posts. I can ignore this, and acknowledge that sometimes I may miss some things and over-react myself provoking that (blame the CFS), but I try to avoid direct put-downs and implied insults in disagreement. A disagreement is not an insult. I really think that given your background and working on a doctorate you should be above personal attacks. Can we stick to the discussion without all the insulting innuendo?]

For the record, I think you are the one who is misreading a study here, namely the WPI study. I realize some parts of that are ambiguous, and some statements after publication about ongoing work with XMRV may be wrongly attributed to the original study. Anyway, what I am saying about HERVs and the WPI methodology is not just my own view, I am also getting this from other researchers back-channel. As I am sure you know, researchers get into factions over issues like this, and if you only talk to one side you only get half the story. So here are some of the points I suspect you may be either missing or ignoring:

1. WPI did not run any culture on samples prior to their PCR testing in the Science study, and they got positives from a very small blood amount. This has been confirmed by private emails from Mikovitz to outside researchers (I have not read those emails but my source is reliable). Also the order of presentation in the Science article implies they did not culture cells prior to PCR. The paper implies that the DNA was extracted directly from PBMC's for PCR. I realize the wording of the original study is a little difficult to interpret, I had to have some parts explained to me so I can not claim to understand this perfectly, but I believe what I have been told by outside experts. Am I talking with the wrong experts as you suggest? I have no way to know. But some statements WPI has made afterwards about culturing apparently apply to their ongoing work and not the Science article, although you and others on the forum seem to be interpreting those statements as meaning they pre-cultured prior to PCR and so must other labs. I have heard only the opposite from outside researchers. Other labs should not have to pre-culture PCR samples. WPI only cultured for antigen and infectivity tests and microscope images. IF WPI did pre-culture before PCR, then that is a major omission in their Science article that invalidates that article, and it should be amended or retracted. But of course that is not the case because as Mikovitz has apparently told people, they did NOT culture before PCR.

2. You raise a good point about the peer reviewers and the expert virologists who have made positive comments about the Science study. However, consider also that neither the peer reviewers nor the expert virologists, nor Judy Mikovitz claim to be experts in CFS. The peer reviewers and expert virologists might not have even known about the connection between CFS and HERVs. In fact the peer reviewers apparently wanted the paper to not even mention CFS. But we know that CFS has a host of very strange medical presentations in the brain, viral immune and detox systems, and that HERVs may be activated in CFS. So is it not possible that their assessment would apply more to ordinary retroviral patients and maybe they have not thought through all the special issues related to CFS in making those comments since they are unfamiliar with our disease? Also, if you are going to refer to experts, be sure to look at both sides. Not all experts are happy with the current XMRV testing and results. Dr Kerr for example has apparently believed at least his study and will not take further funding for XMRV research.

3. My opinion of the European studies is tempered by the fact that WPI did not pre-culture their PCR tests, and if there were ANY real CFS patients in the three cohorts used, then some of those studies should have had some positives. The fact that one UK study did include an antibody test and had some hits (more on the controls) suggests that the MuLV antibody study worked, as it hit something, such as an activated HERV perhaps, or some other antigen it cross-reacts with. If any significant number of those patients had XMRV, there would have been many, many more hits from the MuLV antibody. So why are these studies all negative? Either the cohorts were completely non-specific for CFS, in which case there should have been 2-4% positives, or the tests were incompetent, but they did get hits on control positives so even if they were diluted in strength there should have been some positives, or there was simply no XMRV in the patient population. Remember, WPI got hits on PCR without culturing. So that is why I am not making a big deal of the European studies except what I have already said, the UK studies had low sample volumes and one did not fraction out the Lymphocites further lowering the resolution, and the Dutch study may have had a very weak cohort (we can not know for certain without sample-by-sample background checks since in any study like this the samples may always exceed the diagnostic criteria). But some of those tests were RT-PCR and very sensitive and should have gotten some hits, even without culturing, given the WPI claims.

4. The situation in the UK is awful and I realize that XMRV, if it is true for CFS, could make some change happen there. That is great, but I don't think that issue has any place in the discussion of the science of XMRV. The lack of careful analysis of the WPI claims suggests to me that a bias is at work here on the forum. I want answers as much as anyone, but only correct answers. A false finding that is over-promoted could do tremendous harm to our cause, so I think it is best to wait for more studies before turning XMRV into an advocacy campaign, trying to force CAA to change their cautious stance towards XMRV, and raising patient hopes and expectations. Honestly I wish everyone would just wait, put this on hold, at least until after the CDC study and a few others are released (hopefully by mid summer). Anyway, CFS certainly DOES have a biological root cause, regardless of the outcome of XMRV, this is not the only angle for a solution to the political ME problem in the UK, I believe there is already more than enough evidence that CFS is biological, maybe the CDC and CAA should send a letter of protest to the NHS, and UK ME patients should find some way to have a voice. I would certainly sign on to any petition as a 'friend of the UK ME community' to advocate for change in the NHS position on ME, as I believe most here would, you really are among friends... You have to tackle political problems with better politics.

5. You say I speculate, and sometimes I do, just as you and many others here. So here is my connecting the dots, I think WPI may have inadvertently demonstrated the huge involvement of HERVs in CFS. After all, their 98% positive rate (or whatever it is) is using an antibody type (MuLV) that can sometimes be used to test the presence of HERV activation! That would be huge, and in a political sense would be better for us to have a HERV I think than an exogenous and communicable retrovirus (but that is a separate political issue, of course).

6. You have mentioned repeatedly that without culture the virus can not be found. I question that, particularly given one study that I quoted a few weeks ago that showed dozens of copies of XMRV in every infected cell (sorry have lost that ref, it is back on one of my prior posts). The virus should be found by PCR if it is present in the blood as WPI claims.



Yes, that is an obvious problem with the 'quick and dirty' European studies (anyway that is what some researchers here call that type of study). It shows the low value of ME/CFS to those establishments. Regardless of my other views on the facts, this point is more clear now. To really check the XMRV results the outside labs need to take ME/CFS seriously. That means plan a clean study, carefully build a validation or replication assay and test it thoroughly before even collecting data, collect fresh blood samples, write a balanced report, draw only supportable conclusions, go through hostile peer review, and then give us something useful to talk about. (and yes, I do value hostile peer review, that is the best kind, a good scientist is as happy to be proven wrong as proven right, as both build knowledge, and anyway the important point is to get to the proof, not to be right).



Sorry, I am missing your point here.

HERVs are not completely understood yet, I know there is an issue with their being more active when methylation is failing, as LTR methylation is required to keep them dormant. Also, that HHV is known to activate them, but my point was that WPI used an MuLV antibody for the final step in this test, which is known to cross-react with some HERV classes. Therefore, the transfection may simply be movement of HERV activation triggers between the samples.

Just words Kurt no relevant facts at all.You have no time to"dig out" the data which you claim to have to support your arguments-as yet unseen-but you have time to write a convoluted letter of such length!!