Switzer - Publishes new XMRV varieties, and challenges Coffin's recombination theory

[eric_s]

They say PCR alone is not enough and that you need to use other methods as well to find a good percentage of infections, with PCR you would only find a fraction. They often mention how important culturing is.

I think in these videos she talks about the methodology
http://www.youtube.com/watch?v=maXw3IzSYs4
http://www.youtube.com/watch?v=_paTVFUvZHA
http://www.youtube.com/watch?v=-6xWNVQGPKI

 

[GaryK]

Thanks Eric for the JM youtube videos...I always enjoy her talks on XMRV She is an amazing Scientist!

GaryK

 

[RedRuth]

Thanks I've watched them, she does quickly outline why she thinks her results haven't been replicated

1. Patient selection, though some of the samples from the negative papers used samples that had previously tested positive.
2. PCR conditions, specifically annealing temperature (this is determined by the specificity of the primers). She says there's too much sequence variation to use high annealing temps which is fair enough. Except that there's very little variation in the sequences she's submitted and the only study I checked used the same annealing temp as her group (57C)
3. Culturing, it's actually quite difficult to work out what she did with her cultured cells (as far as I can tell, western blotting and flow cytometry) and why she thinks, for example, the Levy study didn't replicate their results.

She also shows a slide on RT-PCR and IP (immuno precipitation) of blood plasma and suggests that this may be a more sensitive technique than PCR of PBMCs. Though this begs the question of why she didn't put this data in the Science paper - though the IPs are, well, of less than acceptable quality. However this is not what the Switzer paper (that's the subject of the OP) found. All their samples were negative using RT-PCR on plasma.

There is also question mark about the positive controls that the negative studies used and this goes back to why qPCR is a superior technique. For instance the Levy study quantified the sensitivity of their PCR and it was extremely good. The point being that if the assay picks up the control it should pick up positive samples.

BTW, I should say that I think there's some confusion over what scientists mean by replicate. Generally we mean replicate the findings using methods that should give the same results, it doesn't necessarily mean using the exact methods. We always refine techniques as I'm sure the Mikovits group has and some of the groups have used superior methods. TBH if only one group can get a result using an inflexible method then this suggests contamination rather than incompetence on the part of other groups.

Coffin (I think) makes an interesting point, he says that in his opinion XMRV is not worth pursuing but that doesn't rule out a possible viral (maybe MLV but I can't remember) link to ME and that this should be pursued.

On a personal note I think Alter (?) was the only one who retained his dignity.

Anyway those are my thoughts after watching the vids, doing a bit of background reading and taking my personal experience of some of the techniques into account.

 

[Jemal]

Coffin (I think) makes an interesting point, he says that in his opinion XMRV is not worth pursuing but that doesn't rule out a possible viral (maybe MLV but I can't remember) link to ME and that this should be pursued.

Yes, very interesting, but his words are probably empty. He's not going to look for this virus in ME/CFS. Also I am not convinced that XMRV is not worth pursuing.

 

[ukxmrv]

Ruth,

With all due respect you seem to be just repeating opinions. It's not coming across as a fresh scientific mind. Would I be right in thinking that you have never been directly involved with the discovery of a new virus and the work around determining the best methods for detecting it?

I think that scientists are not without common sense. If some groups can detect XMRV and related viri and others can't then the obvious place to check is the difference in methods.

What may be a "superior" method for HIV, may not be the "superior" method for XMRV. We don't know yet what the superior method may be. The BWG is trying to find the best method and if you has read their work you would see that even the day the bloods were processed on made a difference.

This should be a huge red flag for the importance of method.

It doesn't mean that anyone's methods are inflexible and it's contamination - no, it means that for this particular virus then this is the way to do it.

The virus is either in the population or it isn't. If it is in 2-6% of the general population then scientists with the 0/0 testing controls should find it.

The 0/0 studies simply don't make sense unless the virus is not in the general population or their methods are wrong.

Coffin isn't a clinician. He's new to ME and CFS so his opinion is not necessarily worth considering as he lacks the experience. If he thinks that there is a virus then they let him find it - the rest is just posturing to try and convenience patients that he is taking this serious. We need actions not more words.

Alter may have come out with his dignity (your opinion) but he's either right or wrong. If Alter/Lo can find MLV type virus in patients and controls then it's either there or not there.

 

[Cort]

IMO, that part of Switzer's research is the most significant, and if we've interpreted what he's saying correctly it's big news. If he's even suggesting that there may be other ways for XMRV to evolve, and sequences that demonstrate that possibility, and evidence of recombination in this family of viruses, then that is a real blow to Coffin and Paprotka's argument. So I really hope you (or somebody else here) will read that paper and report back - because it really does seem crucial: on the face of it, it has the potential to completely undermine Paprotka and Coffin's contamination theory.

I don't think it necessarily does anything to Coffin's and Paprotka's arguments regarding CFS patients and it won't until somebody finds sequences like Switzer found in those 3 prostate cancer patients in CFS patients. If he's found something unique then good but until the CFS sequences start popping out of the 22RVI phylogenetic tree like XMAS lights it appears that many researchers now are going to assume that the sequences found in CFS are derived from contamination. Researchers simply have to start finding similarly strange sequences in CFS patients for it to make a difference in CFS. Since the WPI must given GenBank their most variable sequences - and they obviously have the biggest store of CFS-related XMRV sequences of anyone - - the fact that that attempt didn't work is troubling. My guess is that we've seen the limits of XMRV variability in the blood in CFS and its very low (Unless an analysis is being done right now on those additions that has positive results.)

The hidden in the tissues question is enticing but that idea won't begin to be studied until a number of labs find XMRV in CFS patients blood (and publish their results). Only then will they begin to look in the tissues. That is the first necessary step.

(Now I will go back into my hole)

 

[ukxmrv]

Cort,

Sad to hear that you are in a hole.

I'm very disappointed with scientists who are basing their assumptions on something like phylogenetic trees. They are not "real" things and prove absolutely nothing.

Phylogenetic trees for anyone who hasn't been involved with them for XMRV, are mathematical models that supposedly show how different things (like viruses) could possibly related to one another.

They do not show how things do in reality relate to one another and they do not prove anything. They are just a way of comparing and guessing how data could possibly be related. Like all mathematical models they suffer from the old "garbage in, garbage out" and there are many different types, and different parameters that can be set up to determine the outcome. A lot of guessing.

Scientists will need to come up with real data based on actual things if they wish to prove that contamination of samples or equipment occurred. I for one, wish that they would either provide the proof or start looking at this problem with more open minds.

Even if they want to argue that phylogenetic trees are "proof" they are still stuck with other scientists who argue that this is in fact proof for the contaminated patients theory.

i.e. that the lack of genetic diversity is because of a common vector

(start)
Once a virus is endogenised, it is forced to follow the evolutionary rate of the host. Since XMRV is integrated in cell-lines the virus evolution is restricted to the host's pace of evolution, and viral descendants have none or minimum sequence diversity. Thus, if a contaminated product, previously cultured in cell-lines, is administered to people then the infections would provide the evolutionary patterns reported by Hue and colleagues.4 If the immunological data reported by Lombardi and colleagues5 are correct, then we need to trace the common source of XMRV and not through human contact

The Lancet Infectious Diseases, Volume 11, Issue 4, Page 264, April 2011
(end)

The scientist arguing "contamination" cannot have it both ways. It seems that they lose credibility by not examining all possibilities.

 

[Cort]

Cort,

Sad to hear that you are in a hole.

I'm very disappointed with scientists who are basing their assumptions on something like phylogenetic trees. They are not "real" things and prove absolutely nothing.

Phylogenetic trees for anyone who hasn't been involved with them for XMRV, are mathematical models that supposedly show how different things (like viruses) could possibly related to one another.

They do not show how things do in reality relate to one another and they do not prove anything. They are just a way of comparing and guessing how data could possibly be related. Like all mathematical models they suffer from the old "garbage in, garbage out" and there are many different types, and different parameters that can be set up to determine the outcome. A lot of guessing.

Scientists will need to come up with real data based on actual things if they wish to prove that contamination of samples or equipment occurred. I for one, wish that they would either provide the proof or start looking at this problem with more open minds.

Even if they want to argue that phylogenetic trees are "proof" they are still stuck with other scientists who argue that this is in fact proof for the contaminated patients theory.

i.e. that the lack of genetic diversity is because of a common vector

(start)
Once a virus is endogenised, it is forced to follow the evolutionary rate of the host. Since XMRV is integrated in cell-lines the virus evolution is restricted to the host's pace of evolution, and viral descendants have none or minimum sequence diversity. Thus, if a contaminated product, previously cultured in cell-lines, is administered to people then the infections would provide the evolutionary patterns reported by Hue and colleagues.4 If the immunological data reported by Lombardi and colleagues5 are correct, then we need to trace the common source of XMRV and not through human contact

The Lancet Infectious Diseases, Volume 11, Issue 4, Page 264, April 2011
(end)

The scientist arguing "contamination" cannot have it both ways. It seems that they lose credibility by not examining all possibilities.

The gene evidence is an important piece but its only part of the overall picture. There is the inability of many labs to find even the background levels of XMRV in the population. It would be interesting to see if any other pathogen has emerged from such a rough road intact. Perhaps there is - my guess is there has not been.

I have the feeling if XMRV is going to turn its going to overturn a good deal of perceived opinion....I went so far as to look up HIV - one of the most variable viruses known and antibodies were found for HIV early on - 25 years ago - and they worked immediately even with such a variable virus...and with XMRV we have an apparently very homogenous virus and now with all our technology the antibody studies aren't working either. They were what supposed to be - what was going to save XMRV. DR. Mikovits said - yes you can argue about the PCR but the antibody tests prove XMRV is real and yet all the other antibody tests are negative...So the research community doesn't know how to do antibody tests either?

I know the WPI is very confident and maybe I place too much faith in the research community but I think somebody should have been able to find it. That is probably the biggest thing for me.... I think that one of the 25-30 labs that came up negative should have been able to find it. Could they all be wrong? Annette said some postive studies will be coming out - that will really be something when that occurs.

 

[currer]

Hi Cort,

HIV has an astronomical replication rate. Check it out on Wiki. It is hard to believe.

I think this would have made HIV easier to find.

Just because XMRV (certainly) does not behave like HIV does not mean it cannot exist. If Ruschetti thinks it is there, I will believe him.
I dont think we should draw parallels with HIV.

I said this today on another post, but I think XMRV causes a latent infection, after its initial spread through the bloodstream, lies low in the tissues as a provirus, and probably induces an auto-immune disease as it cannot be eradicated.
I dont think ME/CFS symptoms are caused directly by virus, but by the body's immune system.

It is early days and researchers are still learning how to find the virus. Switzer found it - not much, but he thought it was there. So it either exists or it does not.

If it exists where did it come from? Its genome suggests that it comes from lab mice not wild mice.
Now we have just learnt that MLVs have been contaminating laboratories (and piossibly staff too) since the seventies.

For me this forms a coherent whole, one that is worth investigation, not to be prematurely dismissed.
In fact its possible origin suggests why some are keen to bury this research.

But I doubt that can happen. So many virologists are fascinated by this virus, more information will continue to emerge.

 

[Cort]

HIV would be easier to find but more difficult to create either antibody or PCR tests for, I would think, - because its so variable. XMRV, on the other hand, is harder to find but should be easy to create tests for because its so homogeneous.

I disagree that some want to bury the research because of its putative origin in a lab. The paper and others suggesting so have been published, some by NIH funded scientists- and that aspect has been freely discussed with the National Cancer Institute even producing a video explaining how they thought that happened. Even after those papers were published there were no media reports that I saw exclaiming "lab creates virus!"; which suggests the media doesn't think that's a hot topic at all.

It appears that yes, the scientific community is interested in this virus - no matter what happens with CFS - and we will undoubtedly learn much more about it over time. Would that they were as interested in ME/CFS!

 

[currer]

Hi Cort,
In Britain the media is strictly controlled on CFS/ME subjects. You would not believe the lies! Going by what you read in the papers does not work here, though I admit things are much better in the US.
The English are very cynical as a result of this, and because historically we have had a more oppressive political ans social system. So my origin shows.

I am very disturbed by the EEC on the science paper. It seems premature to me and in any case not usual as there is no evidence of fraud in the science. Honest mistakes in science are usually left alone. Science progresses by learning from research that is wrong as well as right. So why put pressure on the WPI to withdraw their paper? Doesn't that seem motivated by politics rather than science to you?

We read research papers all the time, but for most journalists a story is not topical for long and they rapidly loose interest or dont inquire too deeply. I think our judgements are worth more than the media's because we think about the subject more.

 

[RustyJ]

I disagree that some want to bury the research because of its putative origin in a lab. The paper and others suggesting so have been published, some by NIH funded scientists- and that aspect has been freely discussed with the National Cancer Institute even producing a video explaining how they thought that happened. Even after those papers were published there were no media reports that I saw exclaiming "lab creates virus!"; which suggests the media doesn't think that's a hot topic at all.

I am surprised at the direction of these statements. The inference that because the media haven't run with the story, there is nothing to worry about, doesn't wash. The media response is controlled by vested interests. This is well documented on both sides of the Atlantic. If the official line is the virus is not dangerous, nor transmissible, then why would the media be interested, unless overwhelming evidence is produced to say otherwise.

GlaxoSmithKline and News International are in collusion. It's a matter of public record. This is fundamentally how business works.

Furthermore, the NIC video does not reassure me. It is a PR exercise in damage control, merely a move to curb any potential hysteria: "We are in control, we know what we are doing!" This is fundamentally how government works. Yet for 50 years they had no idea what MLVs were doing in their labs.

These people have only good in their hearts and our best interests in mind? This is fundamentally not how the world works.

 

[leela]

Brain-fogged non-scientist chiming in here:

The hidden in the tissues question is enticing but that idea won't begin to be studied until a number of labs find XMRV in CFS patients blood (and publish their results). Only then will they begin to look in the tissues. That is the first necessary step.

I think the arguments in this thread really come down to this sad, and to me totally illogical stance (not you, Cort, the general science stance.)

The macaque study clearly showed that the virus showed up only briefly in blood and then retreated quickly to the tissues.
Why oh why can we not find it *first* in tissues, and use that confirmation of infection to then work together as a scientific community
to determine the best high-throughput blood analysis?
I felt that this is what Bob was talking about in his first post about the Switzer paper.

I know I am not a specialist, but this all just seems to be backwards!
Sometimes the trees and the forest both get missed when focused on the microscope, the politics, etc.....

 

[eric_s]

Why do people say other groups can't find the virus? It's not true. As we've pointed out many times, there are at least 5 groups that have been able to find XMRV/MRV.

And i also think it does not make sense to focus on the blood, if a virus might be easier to find in tissues. It seems sensible to me to look where you have the best chance of finding it. We should want to find any virus that is there, not only those that can be found in the blood. But they are looking at other ways than blood tests, we've heard of Silverman and Ruscetti looking at urine samples and De Meirleir looking at stomach tissue.

 

[RedRuth]

Yes, very interesting, but his words are probably empty. He's not going to look for this virus in ME/CFS. Also I am not convinced that XMRV is not worth pursuing.

What I meant was that he clearly thinks there is a viral link to ME but it's probably not XMRV. I assume that means he isn't going to give up looking for the link.

 

[RedRuth]

With all due respect you seem to be just repeating opinions.

My opinions are my own and are based on watching the videos and my extensive experience in molecular biology. I'm not sure if you know how these things work but Mikovits being final author and PI is unlikely to have done any of the work in the Science paper, it's post docs like me (and I assume) Lombardi that do the actual work.

Would I be right in thinking that you have never been directly involved with the discovery of a new virus and the work around determining the best methods for detecting it?

I've said several times that I'm not a retrovirologist I am however in a position to comment on PCR, western blotting and IP. All I've done is listen to what Dr. Mikovits said to a meeting of other retrovirologists and commented on HER list of reasons for why other groups haven't found XMRV. If you check the methods section of the Levy paper then you can see that she's wrong about annealing temperatures. BTW, if you want the opinion of a virus hunter then surely Levy trumps Dr. Mokovits every time.

If some groups can detect XMRV and related viri and others can't then the obvious place to check is the difference in methods.

Or contamination. I would say that one of the biggest problems is that all of the negative studies have been able to pick up the positive control at extremely low copy number. This means there isn't anything wrong with the PCR conditions or the qPCR conditions which leaves sample preparation or patient selection (unlikely given that the virus is supposed to be in the general population)

 

[RedRuth]

The scientist arguing "contamination" cannot have it both ways.

I don't think they are. They're saying the lack of sequences variation is evidence for the contamination theory. They also say that what little variation there is in the sequences put's them as derived from 22RVI As Cort says................ Since the WPI must given GenBank their most variable sequences - and they obviously have the biggest store of CFS-related XMRV sequences of anyone - - the fact that that attempt didn't work is troubling. My guess is that we've seen the limits of XMRV variability in the blood in CFS and its very low (Unless an analysis is being done right now on those additions that has positive results.)

Why do you think they're trying to 'have it both ways'? Perhaps I've misunderstood your argument?

 

[RustyJ]

Or contamination. I would say that one of the biggest problems is that all of the negative studies have been able to pick up the positive control at extremely low copy number. This means there isn't anything wrong with the PCR conditions or the qPCR conditions which leaves sample preparation or patient selection (unlikely given that the virus is supposed to be in the general population)

Not a statement of fact, only opinion.

Redruth, you still have a bit of reading to do. You should, given your experience, know that there are positive controls and there are positive controls. None really used positive controls; they used cloned virus from their own cell lines. At the moment the CDC doesn't even acknowledge the existence of human positives.

The only study that tried to test WPI patients, from samples supplied by Dr Peterson, has big question marks over it, given the personal conflicts of interest involved.

So if the controls are wrong, then the PCR conditions are irrelevant or untested.

Your own statement outlines the paradox, how come the negatives are not picking any positives at all?

And you should also by now be aware that many of these studies are not trying to replicate WPI's assays, but confirm their own, for patent purposes.

 

[RedRuth]

I disagree that some want to bury the research because of its putative origin in a lab. The paper and others suggesting so have been published, some by NIH funded scientists- and that aspect has been freely discussed with the National Cancer Institute even producing a video explaining how they thought that happened. Even after those papers were published there were no media reports that I saw exclaiming "lab creates virus!"; which suggests the media doesn't think that's a hot topic at all.

It appears that yes, the scientific community is interested in this virus - no matter what happens with CFS - and we will undoubtedly learn much more about it over time. Would that they were as interested in ME/CFS!

This is an excellent point. I really don't think conspiracy theories are very helpful here.

 

[RedRuth]

Your own statement outlines the paradox, how come the negatives are not picking any positives at all?

They are, they're picking up the positive controls. The controls they use are perfectly valid because they are the same sequence as the PCR products from the WPI. The point is to have a piece of DNA in the reaction that the primers will bind to and amplify the intervening DNA sequence. This is what they have done, the origin of the positive control is irrelevant. They are testing the PCR conditions ie primer sequence/concentration, dNTPs, etc etc. If they can amplify DNA from the positive control they should be able to amplify DNA of the same sequence (or very similar) from the sample IF the DNA is there.

I'll check what other controls they do.