Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice

[Dusty Miller]

Dusty, I think consulting a specialist on DNA viruses about latency is disingenuous. Their DNA is in episomes outside chromosomes, thus requires independent regulation. Proviral DNA from retroviruses is integrated into chromosomes, which means other epigenetic controls are possible. Epigenetics is a fairly large subject.

I'm still trying to imagine the mental model in play. Do you think ERVs corresponding to simple retroviruses (type I and II) are constantly transcribed? Do you think it is just a fluke that they can sit there quietly for millions of years? Do you think the ratio of proviral DNA to transcribed RNA in infection by simple retroviruses stays approximately constant? Everything I try to imagine seems ridiculous.
I would hate to impute such reasoning to you, so there must be another explanation.

OK, perhaps I have been working with MLVs in culture for too long, where any virus that lands in a nonpermissive site in a chromosome is quickly overrun by new viruses that remain active after integration, such that every cell is turned into a virus factory in short order. Or perhaps I am blinded by the research on insertional activation of oncogenes by MLVs, where one designs the experiment to get continuous virus replication, without which oncogenesis does not occur.

Yes, I do agree that some endogenous retroviruses are latent by the definition I gave earlier, that is, they do not produce virus but are capable of doing so under some conditions. Other endogenous retroviruses, including your example that has been sitting quietly in the genome for millions of years, are no longer latent, but are dead because of multiple mutations and deletions occurring over the years. Many human ERVs fall into this category.

(PLEASE don't respond that I should call these viruses partially dead because parts can be recombined with parts from other ERVs to generate infectious viruses. I know!)

But why argue over these points? I believe what most patients on this site are trying to figure out is what's responsible for their disease. Specifically, in the context of the XMRV story, could it be that some related retrovirus is responsible, and how would this be investigated? I agree that the Onlamoon et al. study indicates that XMRV can persist in monkeys in the absence of detectable virus in blood. Call this latent virus infection or persistent low-level virus replication in cells other than blood cells, it doesn't much matter. What is important is that this finding implies that not finding a virus in a person's blood doesn't mean that that person is not infected, or that virus won't appear sometime later. Following this line of thought, and in consideration of the acute burst of virus replication observed in monkeys infected with XMRV, one needs to assay for virus in blood at multiple time points, especially during early stages of disease. Alternatively, based on the monkey studies, one could look for virus in other tissues where it might be latent (not replicating) and/or persistent (still replicating but perhaps at low levels).

Doesn't this seem more like the discussion we should be having here? (Ignoring the fact that we are way off course with regard to the paper that initiated this thread.)

 

[RustyJ]

For those who are interested and missed it first time round, the Virology blog of Prof. Vincent Racaniello gives an overview of the Macaque XMRV study (Onlamoon, 2011) http://www.virology.ws/2011/02/17/xmmv-infection-of-rhesus-macaques/

Try this: http://www.virology.ws/2011/02/17/xmrv-infection-of-rhesus-macaques/

 

[Dusty Miller]

Dr Miller,

Thanks for answering our questions. I am wondering if you can comment on the phenomenon that is described in this paper:

Suppression of natural killer cell activity by Friend murine leukemia virus. ​

http://www.ncbi.nlm.nih.gov/pubmed/6202923



I have been curious about this ever since the link between XMRV (or really any gamma-retrovirus) was suggested. What is known about this? What is the mechanism and do other gamma-retroviruses (or retroviruses in general) share this ability to suppress NK cell activity? NK cell activity is known to be very low in people with CFS. Its one of the only markers that has been consistent across multiple research studies and appears to also follow the severity of symptoms. Secondary infections that are usually controlled by NK cells also are described as being more active in patients with CFS (ie herpes type viral reactivation) by most doctors who have experience in this illness. I'd be curious your take on this as it would seem that if we could figure out what is causing the shift in NK cell function, it might lead us to the offending pathogen.

I'm worn out here. Hope that someone else will give this a stab.

 

[RustyJ]

OK, perhaps I have been working with MLVs in culture for too long, where any virus that lands in a nonpermissive site in a chromosome is quickly overrun by new viruses that remain active after integration, such that every cell is turned into a virus factory in short order. Or perhaps I am blinded by the research on insertional activation of oncogenes by MLVs, where one designs the experiment to get continuous virus replication, without which oncogenesis does not occur.

Yes, I do agree that some endogenous retroviruses are latent by the definition I gave earlier, that is, they do not produce virus but are capable of doing so under some conditions. Other endogenous retroviruses, including your example that has been sitting quietly in the genome for millions of years, are no longer latent, but are dead because of multiple mutations and deletions occurring over the years. Many human ERVs fall into this category.

(PLEASE don't respond that I should call these viruses partially dead because parts can be recombined with parts from other ERVs to generate infectious viruses. I know!)

But why argue over these points? I believe what most patients on this site are trying to figure out is what's responsible for their disease. Specifically, in the context of the XMRV story, could it be that some related retrovirus is responsible, and how would this be investigated? I agree that the Onlamoon et al. study indicates that XMRV can persist in monkeys in the absence of detectable virus in blood. Call this latent virus infection or persistent low-level virus replication in cells other than blood cells, it doesn't much matter. What is important is that this finding implies that not finding a virus in a person's blood doesn't mean that that person is not infected, or that virus won't appear sometime later. Following this line of thought, and in consideration of the acute burst of virus replication observed in monkeys infected with XMRV, one needs to assay for virus in blood at multiple time points, especially during early stages of disease. Alternatively, based on the monkey studies, one could look for virus in other tissues where it might be latent (not replicating) and/or persistent (still replicating but perhaps at low levels).

Doesn't this seem more like the discussion we should be having here? (Ignoring the fact that we are way off course with regard to the paper that initiated this thread.)

Why argue over these points? Because the paper queried some of the basic conclusions of earlier negative studies. If those studies are invalid, your fall-back argument is that the pathology isn't there - a point you have made a few times.

I would have thought that the issue of retroviral latency is critical to pathology in me/cfs.

While I am pleased that you have found some merit in the work of Onlamoon et al, it concerns me that if researchers, who are purporting to be authoritative on XMRV or retroviruses in general, are unsure about latency, or have discounted it, XMRV aside, then I must ask what else has been glossed over.

The lack of pathology argument is a circular one that has been used to club me/cfs patients over the head. It's one that can, and is, being used to close down any research into biological vectors, because at the moment there is no serious study of disease in me/cfs patients. There are no accepted markers, therefore there is no pathology.

 

[currer]

Following this line of thought, and in consideration of the acute burst of virus replication observed in monkeys infected with XMRV, one needs to assay for virus in blood at multiple time points, especially during early stages of disease. Alternatively, based on the monkey studies, one could look for virus in other tissues where it might be latent (not replicating) and/or persistent (still replicating but perhaps at low levels).

I am so glad to see you accept this, Dr Miller.
Over the last couple of years we have been annoyed by research that makes extravagant claims for non-existence of any virus in studies that do not do this. (Interestingly the need to look for virus at multiple time points has been made by Dr Mikovits)

And how many discussions have we had here talking about the need to look at tissues?
Most of us are so desperate to get our lives back we wouild willingly give up bits of ourselves!

Please remember that if there is a retroviral infection in PWME it will be low level because we survive for years with this disease. It is distressing for us to read research that argues that there can be no pathology because no conspicuous infection can be seen.

 

[Bob]

Why argue over these points? Because the paper queried some of basic conclusions of earlier negative studies.

I would have thought that the issue of retroviral latency is critical to pathology in me/cfs.

It does seem sensible to test Lymphoid tissue in ME patients. It seems that HGRVs are never going to be reliably detected in the blood of patients. But the problem for us, as I see it Rusty, is that until someone carries out an MLV-related study using lymphoid tissue, or possibly a spinal tissue biopsy, looking for the correct MLV-like sequences, using the correct methodology, then we're not going to move forwards in relation to looking for MLV-like viruses in ME patients. But I can't see this sort of research happening. No one is going to get funded for that sort of study, and most retrovirologists have lost interest in HGRVs and ME patients anyway. But if any such HGRV study ends up being negative, and if any part of the methodology isn't to patients' liking, then it would be another wasted study.

I also have a feeling that Lipkin might have carried out some deep sequencing in his XMRV study, and that any MLV-like sequences that may have been detected were found not to be integrated. I'm speculating here, but if this happens then it's one more nail in the coffin.

So where do we go from here?

I think that one interesting study coming up is the Lipkin pathogen study. The latest news is that he is testing spinal fluid. So if any retroviruses are present there, then they should show up, in theory.

I also think that we are likely to continue to learn more from papers like the one at the beginning of this thread. And I think research like that will continue to flourish.

 

[currer]

Does anyone KNOW whether Lipkin did deep sequencing? I heard he did not.
Can anyone say for sure?

I really feel the issue of retroviral involvement in ME must be pursued to the end. And this discussion has only acted to reinforce this belief.

It seems to me that too often research has concluded nothing is there after studies that are incomplete or poorly designed, possibly because the researchers involved are used to writing research proposals for other, more familiar diseases such as cancer and are unfamiliar with ME.
I am grateful that Dr. Mikovits took the time to study ME and think about the disease processes that might be involved.

Incidentally I remember that certain cells in the immune system can present virus directly to neuronal cells without free virus in blood. (I'm really busy with other stuff atm, and shouldn't be here, I just keep getting sucked back.) Havent time to look for a reference, but I am sure someone else can find it. We have been discussing all this in detail over the past couple of years and there are so many interacting factors to take into account before a definitive statement on MRV involvement in CFS/ME can be made.

 

[currer]

And while I am here I will take this opportunity to say I think it a shame that Dr Snyderman's research on his CLL and CFS has not been followed up. Here you have the evidence of blood markers in response to antiretroviral treatment.

"In summary, a new patient with both CFS and B-cell CLL was identified. Infection with a HGRV/MLRV was suggested by the presence of antibodies to MLRV proteins. He also was positive for a T-cell clonal expansion and had elevated cytokine and chemokine levels typical of patients with CFS. With anti-retroviral therapy he showed improvement in his cytokine and chemokine levels, CFS symptoms and hematological parameters. Presumably his improvement was related to the anti-retroviral effects of treatment. The progressive improvement of his ALC, CD19 cells and trisomy 12 clone lasted 9.4 months. Despite continuation of AZT and raltegravir his leukemia relapsed. Interestingly, during relapse both the total B-cell count and the clonal T-cells increased with the rate of increase of the T-cells appearing more rapid. A second response was induced by the addition of the second reverse Transcriptase inhibitor, tenofovir. Both the total lymphocyte count and the clonal T-cells fell with the rate of decrease of the clonal t-cells appearing more rapidly. At the time of this report the second response is ongoing at 14-20 weeks.
These findings are consistent with the importance of reverse transcriptase in the behavior of the patient’s leukemia and CFS and the potential influence of the clonal T-cells on both these processes. It is possible that inhibiting reverse transcriptase decreased proliferation of both the B-cell clones or the effect might be primarily on the clonal T-cells. The rise and fall of cytokines we have documented would be proportional to the absolute number of the clonal T-cells and secondarily could influence the proliferation of the B-cell clone."http://www.x-rx.net/blog/2011/12/update-from-michael-snyderman-md.html

Could the importance of reverse transcriptase in the effects seen be due to HERV activation?

When HERvs are activated how much viral infection is there? Could it explain the effects seen here? Thankyou for giving your time to coming here to look at some of these points with us, Dr Miller.

 

[ukxmrv]

I'm worn out here. Hope that someone else will give this a stab.

Dear Dr Miller,

I'm not surprised that you are worn out. Thank you for the incredible reply to my question about the NZB mouse and the early NZ study. Glad that there are so many new methods now to detect these things.
I may come back with a few questions but I'd like to spend some time and fully digest your reply. Also your replies to other posters here who know much more than I ever will. I've learned a lot from this discussion and hope it continues. Thanks once again.

 

[anciendaze]

Dusty, I'm glad to find we can agree about several things. I was pretty certain no virologist would tell me it wouldn't be fair for a virus to exploit host cell defenses for its own purposes. A problem in this whole story is that tacit assumptions equivalent to such pleas keep cropping up.

You must feel like you have tangled with a buzz saw. You didn't create the situation w.r.t. this illness. The problem is that nobody has corrected some past mistakes. Those "many excellent studies" you cited do include some I regard as fairly good, within a limited scope. Unfortunately, there is a list of studies which looked for a retrovirus in patient cohorts from which those with signs of viral infection were excluded.

How long would it have taken to find HIV in patients without opportunistic infections?

 

[asleep]

I agree that the Onlamoon et al. study indicates that XMRV can persist in monkeys in the absence of detectable virus in blood. Call this latent virus infection or persistent low-level virus replication in cells other than blood cells, it doesn't much matter. What is important is that this finding implies that not finding a virus in a person's blood doesn't mean that that person is not infected, or that virus won't appear sometime later. Following this line of thought, and in consideration of the acute burst of virus replication observed in monkeys infected with XMRV, one needs to assay for virus in blood at multiple time points, especially during early stages of disease. Alternatively, based on the monkey studies, one could look for virus in other tissues where it might be latent (not replicating) and/or persistent (still replicating but perhaps at low levels).

Dr. Miller, I'm glad to see that you agree there is evidence that warrants serious consideration of both a) transient viremia in the blood and b) non-blood viral reservoirs as possible confounding factors in the study of MRVs in ME to date. Given these considerations, I have two questions:

1) From what I know of the pending NIH/Lipkin XMRV study, it is possible (likely?) that blood samples will only be taken from patients at a single time point. It is almost certain that there will be no tissue testing. How is it that some of your colleagues are not only comfortable with but in some cases actively encouraging the idea that this study will be "definitive" or "settle" the issue of MRVs in ME when no study to date, including this very Lipkin study, will have properly accounted for both of these well-characterized issues?

2) One theme that proponents of continued retroviral research in ME have heard repeatedly is that retrovirologists would love nothing more than to discover a new human pathogen, and therefore any doubt expressed by such scientists should be taken at face value because it runs counter to their interests. To me, however, it is perplexing that many of your most vocal colleagues seem very eager to "close shop" on this area of research without ever giving proper experimental or even vocal concern to these rather basic and obvious considerations you mentioned. These considerations have been known and discussed for quite some time, and it's very plausible that much of the negative evidence to date has been skewed by failure to account for them, thereby artificially blocking more fruitful research. Yet, these considerations stand to be ignored entirely. Perhaps you can shed some light on this incongruity?

 

[Mula]

What about Supplemental Fig. S3, A? Doesn't that show that full sequences were detected from each cell line?
http://www.sciencemag.org/content/suppl/2011/05/31/science.1205292.DC1/1205292s.pdf


How do you know that Mula? What is the evidence for that?
The reasons that you gave me earlier were not adequate evidence for your claims, as far as I could work out, as I explained in detail earlier.

Do you mean they haven't used the correct primers?
Surely their primers can be checked against the VP-62 genome?

Actually, I've just checked it myself, and if I use one of their env gene primers:
8r 5’-CTGGATGCTACCGGAGCCC-3’
And run a blast, it comes up with XMRV VP-62 100% identity.

Same with their other env gene primer:
F2r 5’-TTGCAAACAGCAAAAGGCTTTATTGGGAACACGGGT
ACCCGGGCGACTCAGTCTATCGGATGACTGGC-3’

That's also a VP-62 sequence.

See the blast search here:
http://blast.ncbi.nlm.nih.gov/Blast...Options_1SuC4f_1FSM_DTkZxcrQ3qq_23to07_21npY7



I don't understand what you mean here Mula. Could you re-explain?

Fig S3 A lists the primers selected for use on cells, mice and xenografts without evidence that these primers are complimentary to the VP62 virus, which a computer comparison is not able to do. In conjunction with reagents and cycling conditions they could pick up a range of sequences, meaning the red bars only represent unknown sequences.

The gag-pro-pol of sequence 22Rv1/CWR-R1 names the cells it is from.

/cell_line="22Rv1/CWR-R1"

 

[Bob]

Fig S3 A lists the primers selected for use on cells, mice and xenografts without evidence that these primers are complimentary to the VP62 virus, which a computer comparison is not able to do. In conjunction with reagents and cycling conditions they could pick up a range of sequences, meaning the red bars only represent unknown sequences.

So you mean that they rely purely on PCR, and they haven't confirmed their results in any other way?
I agree with you on that point.

I don't know what you mean about being 'complimentary' to VP62.
Do you mean that the primers were not verified by testing them on VP62 positive controls or VP62 clones? So they haven't validated their primers?
I agree with you on that point.

The Paprotka paper is not a conclusive paper, and the methodology is weak. It's just explores a hypothetical model of XMRV recombination.

The gag-pro-pol of sequence 22Rv1/CWR-R1 names the cells it is from.

That doesn't prove your earlier point. You were wrong about that point. They had enough sequences from both 22RV1 and CWR-R1, separately, to say that they had detected XMRV in each. However, it's only a minor issue, compared with the overall weakness of the study, as per the other issues we have discussed. And the env genes were detected, as far as the results of their primers indicate, just as the rest of the genome was detected using primers (all except a section of the LTR region, by the look of it.)

 

[free at last]

Dr. Miller, I'm glad to see that you agree there is evidence that warrants serious consideration of both a) transient viremia in the blood and b) non-blood viral reservoirs as possible confounding factors in the study of MRVs in ME to date. Given these considerations, I have two questions:

1) From what I know of the pending NIH/Lipkin XMRV study, it is possible (likely?) that blood samples will only be taken from patients at a single time point. It is almost certain that there will be no tissue testing. How is it that some of your colleagues are not only comfortable with but in some cases actively encouraging the idea that this study will be "definitive" or "settle" the issue of MRVs in ME when no study to date, including this very Lipkin study, will have properly accounted for both of these well-characterized issues?

2) One theme that proponents of continued retroviral research in ME have heard repeatedly is that retrovirologists would love nothing more than to discover a new human pathogen, and therefore any doubt expressed by such scientists should be taken at face value because it runs counter to their interests. To me, however, it is perplexing that many of your most vocal colleagues seem very eager to "close shop" on this area of research without ever giving proper experimental or even vocal concern to these rather basic and obvious considerations you mentioned. These considerations have been known and discussed for quite some time, and it's very plausible that much of the negative evidence to date has been skewed by failure to account for them, thereby artificially blocking more fruitful research. Yet, these considerations stand to be ignored entirely. Perhaps you can shed some light on this incongruity?

These all seem like fair points to me. But of course if the virus is hard to find in the blood, questions do get asked about how the wpi did find it so well intially. Are we saying ( or sure ? ) that the multiple time draws that Dr Mikovits says they did. are the reasons she had success in the first paper so well. Big problem with this thinking, is the ashford draws ( uk study ) did not use multiple time draws. Yet the results were similar ? But it is a very strange coincidence that she was doing multiple time draws. to detect virus long before evidence surfaced that indeed that is what is needed to detect the virus well. Its all pretty confusing. Always has been ?

 

[acer2000]

I'm worn out here. Hope that someone else will give this a stab.

OK, anyone else want to take a stab at this? Or Dr. Miller when you aren't as wiped out?

Suppression of natural killer cell activity by Friend murine leukemia virus.​

http://www.ncbi.nlm.nih.gov/pubmed/6202923

thanks

 

[currer]

The Paprotka paper is not a conclusive paper, and the methodology is weak. It's just explores a hypothetical model of XMRV recombination.

I agree with you there, Bob. Thanks for all your work dissecting the methodology. I can see that logically Paprotka makes far too many assumptions.
It is disturbing that Paprotka has been so intensively talked up as the final word

The points you make about the ease with which Dr Mikovits finds MRVs are valid, as far as I can see, free-at-last. I remember a lecture where Dr Mikovits talked about the importance of multiple draws because her theoretical model of how MEworked (with the virus hiding in WBC) meant that patients were not always viremic.
We dont know yet why these anomalies exist, and I would like to see the reasons for them elucidated and this is why we are all watching the research so carefully.

Burt at least Dr Mikovits had thought about ME enough to develop a theoretical model, unlike her detractors who are not interested in our disease. And I am concerned by the timing of her silencing so she cannot discuss her work

And yet we are expected to be bowled over by Paprotka..!...as are most of the virological community (or so we are supposed to believe! (How can anyone seriously believe this!)

 

[RustyJ]

You and I understand that Paprotka paper was hypothetical, but it just became fact: This is the damage such papers cause. And those that create such myths are fully cognisant of this process.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir
Ayako Furukawaa, Hideyasu Okamuraa, Ryo Morishitab, c, Satoko Matsunagac, Naohiro Kobayashid, Tsutomu Kodakia, e, Akifumi Takaori-Kondof, Akihide Ryoc, Takashi Nagataa, e, , , Masato Katahiraa, e, g, ,

a Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
b CellFree Sciences Co., Ltd., Ehime Univ. Venture Business Laboratory, Matsuyama 790-8577, Japan
c Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
d Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
e Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
f Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
g CREST, JST, 5-3 Yonban-cho, Chiyoda-ku, Tokyo 102-8666, Japan
Received 14 July 2012. Available online 25 July 2012.


Abstract
Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR.

Five different combinatorially 15N-labeled samples were prepared and backbone resonance assignments were made by applying Otting’s method, with which the amino acid types of the [1H,15N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006).

A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex.

PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions.

This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.

http://www.sciencedirect.com/science/article/pii/S0006291X12013903

 

[Bob]

You and I understand that Paprotka paper was hypothetical, but it just became fact: This is the damage such papers cause. And those that create such myths are fully cognisant of this process.

Unfortunately, exactly the same has happened with the PACE Trial. The false spin put on the results has been misreported as 'fact' in other published studies. It seems that science is sometimes no better than anecdotes being passed around the scientific community, much like village gossip.

 

[asleep]

These all seem like fair points to me. But of course if the virus is hard to find in the blood, questions do get asked about how the wpi did find it so well intially. Are we saying ( or sure ? ) that the multiple time draws that Dr Mikovits says they did. are the reasons she had success in the first paper so well. Big problem with this thinking, is the ashford draws ( uk study ) did not use multiple time draws. Yet the results were similar ? But it is a very strange coincidence that she was doing multiple time draws. to detect virus long before evidence surfaced that indeed that is what is needed to detect the virus well. Its all pretty confusing. Always has been ?

free at last, I think that these are good questions and are deserving of scientific answers. I do not, however, think that they sufficiently answer the questions I posed. In essence, what I'm trying to get at is why scientists--who purportedly love discovering new pathogens--are and have been choosing to ignore clear evidence that may partially if not fully explain much of their failure to find the virus. The key being the selective embracing of evidence in designing studies that control (or fail to control) for confounding variables. If someone were to offer the examples you mention as answers to my questions, it would strike me more as a rationalization or excuse for choosing not to utilize all of the best evidence at hand, especially since no one has bothered to work closely with Mikovits to rigorously follow her methodology.

To put it more succinctly, you cannot appeal to someone else's success as a reason for ignoring evidence if you simultaneously fail to precisely follow their methods. Furthermore, even if you do follow their methods precisely, there's still no scientific justification for ignoring new evidence as it comes in.

As for why Mikovits was able to find the virus so well initially, I can't say for sure. It's certainly possible that she knowingly, accidentally, or even fortuitously did control for these confounding factors in her studies. In fact, there is some evidence to suggest that she was aware of the transient viremia issue. Consider the following quote from her followup methodology paper in Virulence:

In our October 2009 publication, we established XMRV infection in the blood products of our patient population by five different methods. Of these methods, single-round PCR on DNA from peripheral blood mononuclear cells (PBMCs), the least sensitive method, required us to use samples from a subset of chronically ill patients we had observed to have persistent viremia. In Figure 1A of our Science paper, we showed that DNA of 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. We included this figure to demonstrate that nested PCR, which inevitably raises questions of contamination, is not essential to detect XMRV in highly viremic ME/CFS patients. The remaining 90 samples described in the paper exhibited very few XMRV-gag specific PCR products and no env specific PCR products following single round DNA PCR of DNA of unstimulated PBMCs. In contrast, when cDNA was prepared from PBMCs, 67% of the samples exhibited gag products upon nested PCR, though PCR with nested env primers did not result in detectable products from these samples

It would be strange and unnecessary to describe a subset of patients with persistent viremia if you were not aware of another subset of patients with non-persistent (read: transient) viremia. It would be even stranger to devote effort to comparing PCR methods on the very basis of the issues of viremia levels and persistence. So it doesn't seem that unreasonable that their published efforts built on prior observations of the viremia issue.

 

[Sam Carter]

free at last, I think that these are good questions and are deserving of scientific answers. I do not, however, think that they sufficiently answer the questions I posed. In essence, what I'm trying to get at is why scientists--who purportedly love discovering new pathogens--are and have been choosing to ignore clear evidence that may partially if not fully explain much of their failure to find the virus. The key being the selective embracing of evidence in designing studies that control (or fail to control) for confounding variables. If someone were to offer the examples you mention as answers to my questions, it would strike me more as a rationalization or excuse for choosing not to utilize all of the best evidence at hand, especially since no one has bothered to work closely with Mikovits to rigorously follow her methodology.

To put it more succinctly, you cannot appeal to someone else's success as a reason for ignoring evidence if you simultaneously fail to precisely follow their methods. Furthermore, even if you do follow their methods precisely, there's still no scientific justification for ignoring new evidence as it comes in.

As for why Mikovits was able to find the virus so well initially, I can't say for sure. It's certainly possible that she knowingly, accidentally, or even fortuitously did control for these confounding factors in her studies. In fact, there is some evidence to suggest that she was aware of the transient viremia issue. Consider the following quote from her followup methodology paper in Virulence:



It would be strange and unnecessary to describe a subset of patients with persistent viremia if you were not aware of another subset of patients with non-persistent (read: transient) viremia. It would be even stranger to devote effort to comparing PCR methods on the very basis of the issues of viremia levels and persistence. So it doesn't seem that unreasonable that their published efforts built on prior observations of the viremia issue.

It's awfully hard to fault other groups for not following Dr. Mikovits's methodology when that methodology was not properly described in her published work. For instance, the Lombardi paper makes no mention of the need for multiple PCR runs or that 5-azacytidine was used in the production of the (mislabelled) Western blot.