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

[anciendaze]

While we are on the subject of cats again. I'd like an opinion on Infectious Endogenous Retroviruses in Cats and Emergence of Recombinant Viruses.

 

[Bob]

What I mean by "The original findings of XMRV association with prostate cancer and CFS have not been confirmed by others," is just that. Many carefully done studies have failed to confirm the presence of XMRV in patients with prostate cancer or CFS.

Hello Dusty,

Yes, there have been many negative studies, but there has been a positive prostate cancer study published, subsequent to the original:

XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors​

Robert Schlaberga, Daniel J. Choeb, Kristy R. Browna, Harshwardhan M. Thakerb and Ila R. Singh,​

September 8, 2009​

PNAS​

http://www.pnas.org/content/early/2009/09/04/0906922106.abstract​

(Results: XMRV in prostate cancer tissue by IHC = 23%. XMRV in benign tissue controls by IHC = 4%)​

​

​

 

[Dusty Miller]

Hello Dusty,

Yes, there have been many negative studies, but there has been a positive prostate cancer study published, subsequent to the original:

XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors​

Robert Schlaberga, Daniel J. Choeb, Kristy R. Browna, Harshwardhan M. Thakerb and Ila R. Singh,​

September 8, 2009​

PNAS​

http://www.pnas.org/content/early/2009/09/04/0906922106.abstract​

(Results: XMRV in prostate cancer tissue by IHC = 23%. XMRV in benign tissue controls by IHC = 4%)​

​

​

Thank you for being alert and asking good questions. The paper by Schlaberg et al. is fatally flawed and should not have been published. If there was a place on the PNAS website to post comments on papers, I would have done so long ago. These authors make a classic mistake of using polyclonal antibodies in an attempt to detect particular proteins, in this case, XMRV proteins in prostate cancer samples. The problem is that these mixtures of antibodies (polyclonal antibodies) raised against particular proteins often cross-react with other proteins, giving false-positive results. Worse yet, in the case of this paper, was how the authors generated their antibodies. They grew the XMRV virus (derived from the VP62 clone) in LNCaP prostate cancer cells. They then harvested the culture medium from these cells, which contains fetal bovine serum, proteins secreted by the human prostate cancer cells, and XMRV, and filtered this mix to remove things larger than 0.22 micrometers. What they did next is uncertain, but it appears they centrifuged the virus, lysed it using detergent, and injected this preparation into rabbits to make polyclonal antibodies. The problem here is that viruses acquire proteins from the cells in which they are grown, in this case, PROSTATE CANCER CELLS. So, they were making antibodies against XMRV and prostate cancer cell proteins. See the problem? They are then using these mixtures of antibodies in an attempt to detect XMRV in prostate cancer cells! No wonder so many of their patient cancer samples stained positive using these antibodies (23%). And, oh, only 6% of these samples were PCR-positive for XMRV. What, no XMRV DNA and yet the XMRV proteins are being made? Whoops. In short, this paper provides no convincing evidence that XMRV is present in humans.

You may be asking how someone with only a passing knowledge of virology could figure this out. It would be difficult. In this case, the scientists that reviewed the paper appear to have missed the problems! Anyway, suffice it to say that there are problems with all of the papers claiming the presence of XMRV in humans, most less obvious than in this high profile paper published in a top journal, PNAS.

 

[Bob]

Thank you for being alert and asking good questions. The paper by Schlaberg et al. is fatally flawed and should not have been published. If there was a place on the PNAS website to post comments on papers, I would have done so long ago. These authors make a classic mistake of using polyclonal antibodies in an attempt to detect particular proteins, in this case, XMRV proteins in prostate cancer samples. The problem is that these mixtures of antibodies (polyclonal antibodies) raised against particular proteins often cross-react with other proteins, giving false-positive results. Worse yet, in the case of this paper, was how the authors generated their antibodies. They grew the XMRV virus (derived from the VP62 clone) in LNCaP prostate cancer cells. They then harvested the culture medium from these cells, which contains fetal bovine serum, proteins secreted by the human prostate cancer cells, and XMRV, and filtered this mix to remove things larger than 0.22 micrometers. What they did next is uncertain, but it appears they centrifuged the virus, lysed it using detergent, and injected this preparation into rabbits to make polyclonal antibodies. The problem here is that viruses acquire proteins from the cells in which they are grown, in this case, PROSTATE CANCER CELLS. So, they were making antibodies against XMRV and prostate cancer cell proteins. See the problem? They are then using these mixtures of antibodies in an attempt to detect XMRV in prostate cancer cells! No wonder so many of their patient cancer samples stained positive using these antibodies (23%). And, oh, only 6% of these samples were PCR-positive for XMRV. What, no XMRV DNA and yet the XMRV proteins are being made? Whoops. In short, this paper provides no convincing evidence that XMRV is present in humans.

You may be asking how someone with only a passing knowledge of virology could figure this out. It would be difficult. In this case, the scientists that reviewed the paper appear to have missed the problems! Anyway, suffice it to say that there are problems with all of the papers claiming the presence of XMRV in humans, most less obvious than in this high profile paper published in a top journal, PNAS.

Thanks for the helpful info. You seem to have studied the paper in great detail!
I think we would probably all agree that the peer review process should never be relied upon!

 

[Dusty Miller]

Thanks for the helpful info. You seem to have studied the paper in great detail!
I think we probably all agree that the peer review process should never be relied upon!

When the paper came out, I was impressed, until I looked into the details.

 

[barbc56]

When the paper came out, I was impressed, until I looked into the details.

Yes, it seems it's always in the details, eh? Thanks Dr. Miller. Your information is much appreciated.

Barb C.:>)

 

[currer]

"But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"

Dr Miller, the authors of the Zhang paper disagree with you as they warn that

"Laboratories working with xenograft derived human cultures should be aware of the risk of contamination with potentially biohazardous human -tropic mouse viruses....
"Infected cultures usually release large numbers of infectious virions, and intra-laboratory spread of MLV virus to other cell lines maintained in the same facilities may occur, confirming the highly infectious nature of MLV virus. Retroviruses have been associated with multiple diseases including solid and hematologic malignancies, AIDS as well as with non-malignant diseases. The high susceptibility of human cells to infection with XMLV, the high levels of reverse transcriptase activity present in culture supernatant fluids and the demonstrated infectivity of the shed virions suggest that such viruses may present potential biohazards to laboratory personnel involved in cell culture facilities or to those handling human xenografts. In addition, the effects of the integrated provirus or the released virions on the biology of infected tumor cells are unknown. Provirus integration into the genome is not random, and occurs preferentially at transcription start sites, CpG islands, DNase-hypersensitive sites and gene-dense regions, suggesting that provirus integration may influence transcription in the host cell.43 Thus laboratories handling or culturing human xenografts should monitor for the presence of MLV, and should consider monitoring personnel for viral antigens or antibodies to them"

 

[currer]

The Zhang paper also remarks that most scientists working with xenograft cultures were unaware of the contamination problem although it agrees that initial reports about this were published in the seventies.

"However, there are virtually no reports of contamination of xenograft cultures published during the past 15 years and most scientists appear unaware of the contamination problem (authors' observations)"

 

[currer]

With all due respect, Dr Miller, I do not believe that reports of MLV-like viruses detected in human tissues can be dismissed so readily. There are many ways an infection COULD spread into the population and this possiblitiy needs to be properly researched, (not dismissed ) by the scientific community. ( And particularly if MLVs are still being considered as gene vectors for therapeutic purposes.)

 

[jace]

The discussion here seems to be focussing on XMRV, which as we know means a xenotropic virus. But, what was found in patients with ME/CFS were polytropic viruses, both in the October 2009 Science paper and in Alter and Lo's paper, the following summer. I am well aware that these papers have been retracted, but I have yet to be given a convincing reason that relates to the scientific issues rather than political issues.

It is very confusing, for us here on the sidelines, when so many 'definite' statements are made by people on both sides of the argument. For instance, Dr Miller, I wonder what you think of this web page?

It begins:

Missing env region in the 22Rv1 XMRV virus

Several papers such as Paprotka (2011) and Hue (2011) have claimed that the positive results from all studies finding murine leukemia virus-related viruses are the result of contamination from a virus they say was created in a cell line called 22Rv1.

These are cells created from the tumor of a potentially infected prostate cancer patient. They claim that this virus only ever infected other cell lines, is not in lab mice, and is the reason for the positive results.

However, looking at the data placed into Genbank we can see that only onecomplete genome of this virus has been sequenced, but no data on the env gene has been included. Furthermore, the cell line this sequence was taken from is not said to be 22Rv1 but is /cell_line="22Rv1/CWR-R1". What sections of this virus were taken from the 22Rv1 cells alone, and what was from the CWR-R1 cells?

The sequence can be found here
http://www.ncbi.nlm.nih.gov/nuccore/334717372

The page goes on into more detail, with graphics, data and analysis. I would be grateful for your take on this.
The following argument is presented to give you a chance at rebuttal, as well.

The Coffin/Pathak Realtime PCR assay was unable to detect any part of the XMRV genome below a concentration of 2000 copies per 100 cells hence they have absolutely no idea whether there were any env sequences belonging to the XMRV VP-62 genome in the early xenografts

They conveniently did not test the later xenografts with the real time PCR assay. They did not use their XMRV specific gag primer to test the early xenografts.

Finding XMRV in the later xenografts and not the early xenografts using a single round PCR does NOT mean that XMRV was not present. It simply means that the single round PCR assay was not sensitive enough. Testosterone is used in this process which would make XMRV multiply like mad. So it could be way to low a titre for a single round PCR to pick up in an early xenograft but would have increased in titre because of the exposure to testosterone so that the sequences could be readily picked up by single round PCR in the later xenografts

Two competing explanations: dodgy PCR assays or a series of crossovers so unlikely that the odds of them occurring are billions to one against.

Miller and Silverman could have solved this riddle because as if by magic they found the tissue belonging to the patient who was the source of the xenografts. They have Silverman's original PCR tests and his FISH and IHC assays. All they need to do is repeat the tests and if the results are negative we would know that there is no XMRV in that tissue and Coffin was right.

Instead of repeating the details of Silverman's tests that found the virus in prostate tissues they use entirely different ones so no one can truly know whether the virus was present or not.

 

[natasa778]

But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen. For example, human cells can be infected by feline leukemia virus in cell culture, but as noted elsewhere in this thread, feline leukemia virus is not a human pathogen.

Huh, thanks for adding your opinions to debate, but a word of caution here as whatever you say will be preserved for posterity. Absolutist statements have throughout history often turned into big sources of embarrassment.

 

[currer]

"But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"
MLVs have been tested as gene vectors because of their ability to infect every human cell via the XPR1 receptor. The gene vector is presumably injected, thus bypassing the body's natural defences and the fragility of the retroviral envelope.
If contamination with XMLVs is as widespread and unsuspected in laboratories as the Zhang paper indicates, is there a theoretical possibility of such human cell tropic MLVs contaminating cell lines used for the development of vaccines?

Should the scientific community be concerned about this theoretical possibility?

(sorry if this is off-topic for this thread)

 

[ikke2001be]

Hi,

I just ask myself why nobody is talking about anti bodies? I was found pos for these antibodies, and as far as I know, nobody is making antibodies against a lab contaminant. I wonder what's the deal here. Do I, and those few others positive for antibodies against XMRV deserve a place in the guiness book of records then?

Regards,
Ikke

 

[barbc56]

Hi,

I just ask myself why nobody is talking about anti bodies? I was found pos for these antibodies, and as far as I know, nobody is making antibodies against a lab contaminant. I wonder what's the deal here. Do I, and those few others positive for antibodies against XMRV deserve a place in the guiness book of records then?

Regards,
Ikke

If I remember correctly, it's because of cross reactivity. The antibodies weren't necessarily reacting to XMRV. This is a good example of the importance of the details that we lay people often miss.

The ability of an antibody to react with or bind an antigen that did not stimulate its production.

http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-C/cross-reactivity.html

Barb C.:>)

 

[Dusty Miller]

"But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"

Dr Miller, the authors of the Zhang paper disagree with you as they warn that

"Laboratories working with xenograft derived human cultures should be aware of the risk of contamination with potentially biohazardous human -tropic mouse viruses....
"Infected cultures usually release large numbers of infectious virions, and intra-laboratory spread of MLV virus to other cell lines maintained in the same facilities may occur, confirming the highly infectious nature of MLV virus. Retroviruses have been associated with multiple diseases including solid and hematologic malignancies, AIDS as well as with non-malignant diseases. The high susceptibility of human cells to infection with XMLV, the high levels of reverse transcriptase activity present in culture supernatant fluids and the demonstrated infectivity of the shed virions suggest that such viruses may present potential biohazards to laboratory personnel involved in cell culture facilities or to those handling human xenografts. In addition, the effects of the integrated provirus or the released virions on the biology of infected tumor cells are unknown. Provirus integration into the genome is not random, and occurs preferentially at transcription start sites, CpG islands, DNase-hypersensitive sites and gene-dense regions, suggesting that provirus integration may influence transcription in the host cell.43 Thus laboratories handling or culturing human xenografts should monitor for the presence of MLV, and should consider monitoring personnel for viral antigens or antibodies to them"

A quote from the last paragraph of a paper from my lab (Knouf et al., J Virol 83:7353, 2009):

"Finally, production of XMRV by 22Rv1 and potentially other prostate cancer cell lines should be carefully considered
from the standpoint of possible virus transmission to laboratory personnel, to other cells cultured in parallel, and as a confounding factor in the interpretation of experimental results."

I agree that scientists should be careful when working with viruses, especially those that can infect human cells. When we first found XMRV in 22Rv1 cells, I emailed American Type Culture Collection (ATCC) personnel and asked them to increase the biosafety level for this cell line, which they did, due to the potential hazard associated with this virus. The ATCC provides characterized cell lines to scientists worldwide. When I work with XMRV I am careful, but not as careful as I would be when working with HIV, a known human pathogen. But, potential hazard is not a known hazard. One can't call XMRV a human pathogen without data that it causes disease in humans. So far, such data is lacking.

 

[Dusty Miller]

With all due respect, Dr Miller, I do not believe that reports of MLV-like viruses detected in human tissues can be dismissed so readily. There are many ways an infection COULD spread into the population and this possiblitiy needs to be properly researched, (not dismissed ) by the scientific community. ( And particularly if MLVs are still being considered as gene vectors for therapeutic purposes.)

With all due respect, the scientific community has not ignored the possibility that retroviruses like XMRV might spread in the human population, and much research has been done to detect such retroviruses in humans. When retroviruses were first found to cause cancer in animals, it was obvious to look for such associations in humans. Part of the excitement generated by the Urisman et al. paper on a potential role for XMRV in prostate cancer was because it would vindicate the huge effort spent looking for such viruses with little success.

With regard to the use of retroviruses in gene therapy, a large amount of effort has been devoted to testing the safety of these vectors. So far, the appearance of replication-competent recombinant retroviruses has not been a problem, but cancer resulting from insertion of the therapeutic vector near cellular oncogenes has been detected in multiple gene therapy recipients.

 

[Dusty Miller]

"But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen."

Huh, thanks for adding your opinions to debate, but a word of caution here as whatever you say will be preserved for posterity. Absolutist statements have throughout history often turned into big sources of embarrassment.

I am simply making a logical statement here. A threat of rain doesn't imply that it will rain.

 

[natasa778]

"But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"
MLVs have been tested as gene vectors because of their ability to infect every human cell via the XPR1 receptor. The gene vector is presumably injected, thus bypassing the body's natural defences and the fragility of the retroviral envelope.
If contamination with XMLVs is as widespread and unsuspected in laboratories as the Zhang paper indicates, is there a theoretical possibility of such human cell tropic MLVs contaminating cell lines used for the development of vaccines?

Should the scientific community be concerned about this theoretical possibility?

(sorry if this is off-topic for this thread)

And would most of them be willing to discuss it openly, in case theoretical possibility is misinterpreted ;-)

 

[ikke2001be]

OK, cross reactivity, but with what? As far as I have read about antigens, they are pretty specific to the pathogen. So if there is cross reaction, doed this mean that the pathogen is "of the same family" as xmrv, or can this happen with completly different pathogens as well?

Thanks for your clarification,

Ikke

 

[Dusty Miller]

However, looking at the data placed into Genbank we can see that only one complete genome of this virus has been sequenced, but no data on the env gene has been included. Furthermore, the cell line this sequence was taken from is not said to be 22Rv1 but is /cell_line="22Rv1/CWR-R1". What sections of this virus were taken from the 22Rv1 cells alone, and what was from the CWR-R1 cells?

Regarding the apparent lack of an env gene in the 22Rv1 virus sequence, this is simply a problem with the annotation of this sequence. That is, someone left out a description of the Env protein, even though an open reading frame from which the Env protein is translated is clearly present in the sequence. Do you really think that all of the retrovirologists working on XMRV would have missed the absence of an env gene in their flagship virus?

Regarding the CWR-R1 designation, this is another name, as is 22Rv1, for cells derived from a particular human prostate tumor. Cells derived from this tumor were initially called CWR22, but as derivatives with different properties were isolated, they were given different names. The replication-competent retrovirus found in both CWR-R1 and 22Rv1 are identical, and thus, both names are included in the sequence description. The viruses from both cell lines have been sequenced multiple times, and their sequences match, therefore the viruses from these two cell lines are considered to be the same.