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

There have been a couple of studies (one paper & one abstract) recently published by William Switzer of the CDC, which have a significant impact on the XMRV contamination debate.

These studies have been discussed on the forum before, but I think that the discussions may have been buried in long threads, so I thought it might be helpful to repost the information here.

Here are details of the two studies...


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No Association of Xenotropic Murine Leukemia Virus-Related Viruses with Prostate Cancer
William M. Switzer, Hongwei Jia, HaoQiang Zheng, Shaohua Tang, Walid Heneine
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019065#abstract0

Although Switzer concluded that there is no association between XMRV and prostate cancer, based on the findings of this study, he did detect XMRV in the tissue of 3 prostate cancer samples, but he could not detect XMRV in the blood of those XMRV-positive patients. He concluded that further investigation of XMRV is needed.

"Sequence analysis of the PCR-positive specimens was highly informative because it confirmed that all three specimens were XMRV-related. Also, the finding of a viral strain in three prostate cancer patients that is distinct from the XMRV seen in previous studies is significant and demonstrates a broader viral diversity. This would be an expected result consistent with virus evolution during spread and persistence."

The significance of Switzer's study is...

1. Switzer confirms that XMRV is a real wild human virus.

2. Switzer concludes that the genetic variation is consistent with normal human infection.

3. Switzer admits that he cannot detect XMRV in the blood of XMRV-positive prostate cancer patients, using established methodologies. This has relevance for all of the other negative XMRV studies.

4. Switzer confirms that his findings not due to contamination.


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The following two abstracts look like they are based on the same study...

Extensive Genetic Recombination in the XMRV Genome
William Switzer, W Heneine1, M Prosperi, and M Salemi
Presented at the 18th Conference on Retroviruses and Opportunistic Infections (CROI) 2011
http://retroconference.org/AbstractSearch/Default.aspx?Conf=20&Abs=40126

Murine leukemia viruses (MuLV) and Xenotropic MuLV-related viruses exhibit inter-tropic complex recombination patterns
Mattia C F Prosperi , William M Switzer, Walid Heneine and Marco Salemi
6 June 2011
Presented at the '15th International Conference on Human Retroviruses: HTLV and Related Viruses'
http://www.retrovirology.com/content/8/S1/A235

Conclusion:
"Given the evidence of inter-tropic recombination in MuLV, detection and classification of recombination in XMRV using different MuLV tropism prototypes should be interpreted with caution ... These results suggest that identification of parental strains of the potential recombinants is difficult and that recombination in the highly genetically related MuLV have been occurring for some time."

The Paprotka et al. paper (i.e. Paprotka, Coffin and Pathak) ruled out everything for XMRV other than a recombination event happening in the specific cell line that they studied. This CDC/Switzer study points out that, contrary to John Coffin's conclusions in the Paprotka et al. study, there are a myriad of ways that XMRV could potentially have been created by a recombination event, and that the prostate cancer cell line might just be one example of many possible recombination events. Switzer draws very different conclusions to Paprotka et al., and directly challenges their study's conclusion of a 'one in a trillion' chance of a recombination event.

The significance of this paper is that Switzer directly challenges, Coffin's theories about the recombination history of XMRV. Switzer appears to be saying that Coffin's 'one in a trillion' conclusion is mistaken, and that there is a vast potential for possible recombination events to have happened to create XMRV.

This study hasn't been published yet, so it is premature to make any definite conclusions from it, but taken together with the published Switzer study, above, the two studies challenge most aspects of the Paprotka et al. (e.g. Paprotka, Coffin and Pathak) recombination study.


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Switzer's sequences published in genbank:

Division of HIV/AIDS Prevention, Ceners for Disease Control and Prevention.
Switzer,W.M., Jia,H., Zheng,H., Tang,S. and Heneine,W.
10th May 2011

Xenotropic MuLV-related virus isolate 5935 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HQ116790.1

Xenotropic MuLV-related virus isolate 5956 gag protein (gag) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003612.1

Xenotropic MuLV-related virus isolate 6203 envelope (env) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003611.1

Xenotropic MuLV-related virus isolate 5956 envelope (env) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003610.1

Xenotropic MuLV-related virus isolate 6203 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003609.1

Xenotropic MuLV-related virus isolate 5956 polymerase (pol) gene, partial cds
http://www.ncbi.nlm.nih.gov/nuccore/HM003608.1


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John Coffin's recombination paper:

Recombinant Origin of the Retrovirus XMRV
Tobias Paprotka, Krista A. Delviks-Frankenberry, Oya Cingz, Anthony Martinez, Hsing-Jien Kung, Clifford G. Tepper, Wei-Shau Hu, Matthew J. Fivash Jr., John M. Coffin, Vinay K. Pathak
31 May 2011
Sciencexpress
http://www.sciencemag.org/content/early/2011/05/31/science.1205292.full.pdf


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Very interesting thanks Bob - even my school biology tells me viral evolution is such an obvious player in infections.

Thank you, Bob, for this thread and for all the information you provide and for your informed opinions on the significance of research results.

greeeeeeeeat stuff. thanks for posting this again!!!! very helpful indeed.

so now what? does coffin respond?

Interesting! Thank you for brigning this to our attention.

What exactly do you mean by that?

Hi RedRuth. I would be curious to know if you or people you work with have conducted XMRV studies?

It's all in the published paper RedRuth.

Switzer detected XMRV in the tissues of 3 prostate cancer patients, and confirmed that this was not due to contamination (because he carried out contamination tests and there was sequence variation indicative of human infection.) But he was unable to detect XMRV in blood samples from the same patients. So Switzer could only detect XMRV in the prostate tissue samples, and not in blood samples. He suggested that this was due to low copy numbers in the blood (i.e. he used an inadequate or inappropriate methodology.)

The relevance for the other negative XMRV studies, is that if Switzer is unable to detect XMRV in the blood of confirmed XMRV-positive patients, using established methodologies, due to low copy numbers (his conclusion), then this may partly or fully explain why so many other researchers are unable to detect XMRV using established methodologies.

Judy Mikovits carefully selected her patients such that she thought they showed signs of viral infection. So cohorts may also play a role in how high the patients' XMRV blood titres are.

No, not at all, I'm not a retrovirologist, I work in a membrane protein trafficking lab. However I do work on membrane microdomains (lipid rafts) and because of the importance of these domains in retroviral entry and exit I am doing some work with HIV accessory proteins.

Yes, I've read it. I'm just not sure what you mean in some of your analysis. You say

Where does he/she suggest this was due to low copy number or inadequate methodology? I think you may be misunderstanding what they have tested for. There are three techniques here

1. PCR - test for DNA
2. Reverse Transcriptase PCR - test for RNA
3. Western Blotting - test for Protein

As far as I can tell they only did PCR on the tissue samples NOT the blood samples. They used RT-PCR (to test for RNA) on the blood samples to look for viremia. His conclusion is The finding of undetectable antibodies and viremia in all three patients may reflect latent infection And in the discussion The absence of antibodies and plasma viremia in these three patients is noteworthy because it may reflect sequestered or latent infections.

Have I missed a reference to inadequate methodology?

I haven't re-read the paper yet, to check the answers to your questions.
Going from memory (which isn't reliable), I think he does make a specific reference to low copy numbers, but even if he doesn't, your own quote says the same thing...
Where he says "The finding of undetectable antibodies and viremia in all three patients may reflect latent infection" is exactly the same as saying "may reflect low copy numbers".
'Inadequate methodology' is my own conclusion, as it seems clear that if you are unable to detect a virus because of low copy numbers then you have an inadequate or inappropriate methodology. That's just pure logic.
If the virus doesn't exist in the blood at all, then you have an inappropriate methodology if you are attempting to determine if someone is XMRV positive.
I acknowledge that Switzer was not attempting to determine XMRV status by using this method, but my point is that if Switzer can't detect XMRV in the blood of confirmed XMRV positive patients, using his methodology, then it is an indication of the difficulty of doing so with other methodologies.

low or latent viruses might be enough to cause a type of encephalitis. They might not be able to distinguish between low and latent or like herpes viruses how they dont/cant tell the difference between latent and recent infections.
I have also thought that even if it was just ebv and it reactivated once a year and then went latent we would feel crappy all year from post viral fatigue and then it would restart again when it reactivates before we recover again. The same could happen with xmrv, but im only speculating but if Coffin can do it, so can I, lol.

cheers!!!

I just posted a thesis that talks about detecting XMRV in the urine of prostate cancer patients. They are looking in urine now, because it's difficult to detect in blood I guess.
see: http://forums.phoenixrising.me/show...-XMRV-As-An-Indicator-Of-Prostate-Cancer-Risk

Yes but copy number and titre are two different things here. When he talks about copy number he's referring to the XMRV DNA, titre relates to virus particles. One copy of a virus can produce multiple (10s, 100s) of virus particles. Table 2 explains what tests were done on which samples. The n(nested)PCR (the test for virus DNA) was only done on tissue samples NOT blood. I really don't see what bearing this part of the paper has on the original Lombardi paper or subsequent negative studies.

The point he's making is that they can't detect virus RNA or Protein in the blood of the patients, virus RNA or protein would indicate the presence of virus particles ie exogenous virus. He also makes the point that viral DNA integration into the prostate tissue isn't the cause of the cancer.

I don't quite understand what point you are making RedRuth.

Switzer carried out PCR on blood samples and he could not detect XMRV in the blood of known infected prostate cancer patients, and he suggested that this was due to low copy numbers in the blood.

The point that I was making is that, whatever the methodology used in other negative studies, Switzer demonstrated the difficulty of detecting XMRV in the blood, and gave us a possible reason why so many studies are having such difficulty detecting XMRV in the blood using established methodologies.

I know that Lombardi et al detected some XMRV in blood samples using single pass PCR, but they used a very specific methodology, and a very specific cohort of patient who they suspected of having high viremia.

Does that address your questions?

You're not being clear about what you mean, when you say 'XMRV' do you mean DNA, RNA or protein becasue all 3 were tested for. Switzer carried out nested PCR to find viral DNA in prostate tissue and suggested that the virus was low copy number in the tissue. They didn't look for viral DNA in the blood and didn't suggest that copy number was the cause of them not seeing viral RNA or protein in the blood, they suggest latency explains the lack of viremia. The Lombardi paper and presumably all the negative papers looked for viral DNA in blood. This is not what Switzer did so direct comparisons aren't valid.

See my point above, Switzer didn't look for DNA in blood.

Single pass?

EDIT: My point is that I think you're getting confused over the different techniques and what they're used for. The interesting/relevant part of the paper from the POV of XMRV/CFS is the phylogenetics stuff in my opinion

Hello RedRuth, would you mind explaining what these 'virus particles' are from molecular point of view and how they originate? Thank you.

I'll try but I'm not a great writer. Retroviruses like XMRV and HIV are basically just a short peice (or 2) of RNA in an envelope ( host cell membrane and virus protein). When this type of virus infects a cell it's RNA genome is made into DNA and incorporates into the host genome. From there multiple copies of the virus genome can be made, then exported from the cell as an infective enveloped virus - this is how one virus can make multiple copies of itself (using the host cell machinery) I've seen images (electrom micrographs) of 10s of HIV particles budding from an infected cell. Does that help?

The image I was thinking of is here http://tracingresistance.blogspot.com/2010/03/restriction-factor-tetherin-not-so-fast.html

Excerpts from the published paper:

"The absence of antibodies and plasma viremia in these three patients is noteworthy because it may reflect sequestered or latent infections."

"Our results are also consistent with those seen recently in macaques experimentally infected with XMRV in which tissues at necropsy are PCR-positive but viremia and detection of provirus in PBMCs disappear quickly, followed by loss of antibody detection."

The above sentences equate to "low copy numbers", or zero copy-numbers. (Absence of plasma viremia = low copy numbers.)

I don't think that I made direct comparisons with other papers, did I?
I merely said that Switzer's results have "relevance for all of the other negative XMRV studies."

I don't think that you have read my opening post carefully, and I can't work out what point you are making when talking about the different methodologies. Switzer looked for viremia in the blood, and he could not detect it in samples from known XMRV positive patients, due to low copy numbers. My point is almost as simple as that.

I agree that the phylogenetics are interesting.

Which would only be relevant to the Lombardi study and all the negative studies if they had looked for virus DNA in the patients blood. But they didn't. That's my point. Also when Switzer talks about copy number he means cells infected with virus DNA, NOT virus particles, XMRV is a retrovirus (RNA virus) you can't test for virus particles with nested PCR (DNA). This paper basically says that the virus is present but it's not doing anything (latent).

They are. Presumably the two papers where in review/publication at the same time so they don't really address each other.