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

[anciendaze]

Ancientdaze, I just want to clear up something you said in your post. You may have meant this but people can not catch feline leukemia from cats.

Didn't mean to imply such. I'm reasoning by analogy with known examples of retrovirus transmission within mammalian species. Besides, I don't know anyone who drinks cat's milk except kittens.

Thinking about the problem of transmission from the standpoint of the virus caused me to concentrate on what may seem unlikely requirements for transmission. Transmission of HIV to infants reveals that this is still a young pathogen, poorly adapted to our species. Active HIV infection in an infant makes that infant unlikely to survive until sexual maturity, when the infection is most likely to be passed on. If the virus were intelligent this would be called a major strategic blunder.

A well-adapted pathogen would never kill an individual host before infection is passed on. The ideal strategy for such a pathogen would be to transmit an infection when a neonate is immunologically naive which would remain latent until sexual maturity. It would infiltrate the immune system without provoking a strong response. I simply asked myself if this were possible. Published research gave me answers, it also revealed that a virus using such a strategy would be more likely to escape detection. Every link in the chain is not solid, yet.

This can be criticized as speculative. I never called the hypothesis anything else, though it rests on facts. It has served to guide me to interesting discoveries within published literature. I'll drop it when and if it stops being productive.

 

[Bob]

Didn't mean to imply such. I'm reasoning by analogy with known examples of retrovirus transmission within mammalian species. Besides, I don't know anyone who drinks cat's milk except kittens.

Your reasoning was very clear, anciendaze, and it was very clear that you weren't implying anything about humans catching cat viruses.

 

[barbc56]

Your reasoning was very clear, anciendaze, and it was very clear that you weren't implying anything about humans catching cat viruses.

Yeah, it probably was, but my brain is on overloaded after a week long trip. However, many people do think you can catch feline leukemia and wanted to make sure that is what you were saying.

Barb C.:>)

 

[barbc56]

Has anyone read the full paper? It appears that there's a lot of speculation here which may or may not be correct and can be interpreted several ways. Nothing wrong with speculation per se but I just like to get as much information as possible.

I googled this paper and all I got was the abstract and the o'keefe blog and I'm not sure what people are speculating here is in sync with what she is saying. It may very well be.

in answer to your first question, I would have to say yes, because how can we know for sure they aren't there if we can't detect them. It really is a matter of probability

From what I read above, I get the feeling that she is talking about a relative young retrovirus but is also saying that probability is a factor when determinating this. Anything is possible but is this probable? She is unclear about this and I would think the probability of an event happening is an important piece of information.

recent could be considered a few million years (its all relative). In this case, to be more specific, we might have expected that samples from different mouse strains and cell lines and humans would be more similar to other sequences from the same source - ie the preXMRV-2 sequences from humans would be more similar to each other than they are to a wild-mouse caught in Germany. And like-wise, sequences from mice of a particular strain would be more similar to each other and separate out on a phylogenetic tree. In addition, not all wild-mice have the virus, again suggesting if it were truly old, then more of them would be positive.

Once again I think there are several interprtations of the above. She is saying there should be more genetic diversity.

I would like to see what scientist have to say about this. My first question would be does this study really refute previous research.

 

[Bob]

Has anyone read the full paper?

I haven't been able to access it.

It appears that there's a lot of speculation here which may or may not be correct and can be interpreted several ways. Nothing wrong with speculation per se but I just like to get as much information as possible.

Agreed. But there is some helpful info in the abstract.

I googled this paper and all I got was the abstract and the o'keefe blog and I'm not sure what people are speculating here is in sync with what she is saying. It may very well be.

I've been basing my interpretation of the Mayer paper on the abstract. But you're right, we need to read the full paper to fully understand it.

From what I read above, I get the feeling that she is talking about a relative young retrovirus but is also saying that probability is a factor when determinating this. Anything is possible but is this probable? She is unclear about this and I would think the probability of an event happening is an important piece of information.

They do seem to have detected PreXMRV-2-like gag genes in the wild.
I haven't read the discussion thread that you are quoting from, but any discussion relating to probabilities would probably be in relation to how and where a virus was created: because it's not possible to determine exactly when or how a virus was created. And also, what relationship a virus has with another virus, specifically the relationship between the new gag sequences and the PreXMRV-2 gag gene.

So, what is the relationship between the new sequences to those in the Paprotka paper?

But I'm not sure if the answer to that question matters, since PreXMRV-2 was not detected in the xenografts in the Paprotka paper anyway. And definitely not the gag gene. Only a tiny snippet of the LTR region was detected, but that does not conclusively indicate that PreXMRV-2 was present, as far as my understanding goes.

The gag gene for PreXMRV-2 was only detected as gDNA in the mouse genomes that might have been used for the xenografts.

My first question would be does this study really refute previous research.

The Paprotka study was not conclusive, but was based on many assumptions. The Paprotka paper seems to describe quite a good hypothetical model, and maybe a convincing model for some people, but it was not a conclusive one. Their conclusion that PreXMRV-1 combined with PreXMRV-2 to create XMRV is purely based on assumptions, probabilities and deductions. For example, they haven't actually detected any active virus, or viral RNA, for either of the two pre-viruses, as far as I can see. And they didn't find PreXMRV-2 gDNA in any of the xenograpfts. They only found a tiny snippet of gDNA that they assume relates to PreXMRV-2, but not even a gag, pol or env gene. Even if they had found the full sequence of PreXMRV-2 (and they say it was probably present in some of the mice used in the xenografts), it is still quite a jump to assume that they know how XMRV was created, because there are so many possibilities, as was explained in a published paper that refutes the Paprotka model (I can't remember which one, but there was one that downplayed the certainty of Paprotka, and discussed the possibilities of recombination events).

I think that the new finding of PreXMRV-2-like gag genes in the wild does give more credibility to theories other than the Paprotka model:
Firstly that PreXMRV-1 could also be present in the wild (if we don't already know this?)
Secondly, that XMRV-like viruses would have had far more of a chance to be created in the wild than in an isolated cell line, if PreXMRV-2 is in the wild.
Thirdly, that an unknown XMRV-like virus could have infected the cell line from an external source.
Or fourthly, that XMRV could have been created in the wild, and that's how it entered the cell line.
(XMRV, or an XMRV-like virus, could yet prove to be a mouse virus or a human virus.)

But like I said, Paprotka was a hypothetical model of the creation of XMRV, and was not conclusive.
And we do need full access to the new paper.

 

[barbc56]

Does age of xmrv refute that it is still a contaminant or background noise that has nothing to do with illness?

Barb C.:>)

 

[anciendaze]

The full paper has not been published, but the accepted draft is available for a price. You can add it to the research archive here if this has not already been done. I read it via another forum.

The reason this virus species is considered young is that it has not even completely spread to the entire population they sampled. It also shows a great deal of variation, which is typical of a virus which is still adapting to a host species. It has been around long enough to leave some fragments as (defective) endogenous retroviruses, but the wild type is not fully represented by these. While "young in an evolutionary sense" can mean millions of years, the evidence so far, plus the rate at which mice evolve, causes me to put the age much lower, at least in the population sampled.

If these were simply ERVs, it would be possible to fit them into some model showing descent from a common ancestor. The data appear to violate that assumption. Horizontal transmission of genetic information is taking place.

The fact that they found the 24 nucleotide deletion characteristic of XMRV in some, but not all, mice, including wild mice, argues that this is not simply a laboratory artifact from a single source. Some gag subsequences they found were not in the XMRV sequence. Contamination by XMRV would not supply them. There is a great deal more to be learned.

I can accept that Paprotka found evidence of a single recombination event, but I have never thought that the pieces going in were all passive ERVs. Some exogenous virus had to be there to start the process, and this virus had to be able to infect the mouse cells carrying the preXMRV1 ERV. There are also parts of XMRV not reflected in either of the two designated components. I want to know where they originated.

 

[ukxmrv]

I've read the full draft paper and find the discussion here is spot on.

Thanks for Anciendaze, Bob and everyone else for the comments and questions.

Barb, if you have a read back through some of the earlier posts on this thread it may become a little clearer. You may also like to reread the Patrotka paper. Nothing is certain here as always but there are questions.

 

[barbc56]

I've read the full draft paper and find the discussion here is spot on.

Thanks for Anciendaze, Bob and everyone else for the comments and questions.

Barb, if you have a read back through some of the earlier posts on this thread it may become a little clearer. You may also like to reread the Patrotka paper. Nothing is certain here as always but there are questions.

You have missed the point. I'm not sure if anyone here, including myself, knows what this study really means as we simply do not have all the facts.

What isn't clear is why we haven't heard from scientist saying this negates other studies that show xmrv is a contaminant or that if the age of the PreXMRV-2 gag sequence is even important. I would think if it does we would be hearing from some of the more credible scientist but I can't find this information, so we don't know if they are dismissing this study or implying it doesn't really change things. I have no idea.

As I said there is nothing wrong with speculation and that is why I am reading this thread to get a feel for what people are thinking as well as information. Unfortunately, speculation sometimes ends up as being taken as fact or is biased towards a certain theory where the facts are cherry picked to fit to that theory.

It's so easy to become an "armchair retrovirologist" and think we understand when in reality it takes years and years of training, experience and education.

I am not saying that is happening here or we should stop this speculation, but I would like to get a better handle on the implications of this study.

The O'Keff blog as well as the abstract can be interpreted several different ways.

Bottom line? We need more information.
Barb C.:>)

 

[Ecoclimber]

Just answering my own question, as none are known yet but still it seems XPR1 is very important to cell function

from http://labs.fhcrc.org/miller/research.html

In later studies we focused on how XMRV might be involved in CFS, and found that XMRV could kill neuroblastoma cells, indicating a neurotoxic activity of XMRV that might be involved in CFS. This toxic effect was mediated by interaction of the virus with its cell-entry receptor, Xpr1, which we recently found was an atypical G-protein-coupled receptor that regulated cAMP levels in the neuroblastoma cells. XMRV infection blocked Xpr1-mediated signaling resulting in reduced cAMP levels and cell death.​

​

... However, we continue to study the role of Xpr1 in cell biology, and are looking for possible natural ligands that might regulate Xpr1 activity. Xpr1 orthologs are present in many organisms, including plants, animals and fungi, and XMRV has provided a window into what may be an important regulatory pathway in a wide range of living things.​

just answering my own question, as none are known yet but still it seems XPR1 is very important to cell function
from http://labs.fhcrc.org/miller/research.html
... and still mice sacrificed it!
btw DM might want to rethink the bit on the origin of XMRV in the light of new data. Just saying ...

I (Miller) will clarify the inaccuracy in post 28 that refers to my work. Mice do have an Xpr1 gene that is expressed in most mouse tissues. One can verify that mice have an Xpr1 gene by doing a blast search for sequences similar to the human Xpr1 cDNA, GenBank accession NM_004736.3 . A little way down the list is GenBank accession NM_011273.2 , the Mus musculus xenotropic and polytropic retrovirus receptor. But the key point is that while Xpr1 genes present in many organisms are related and are therefore all called "Xpr1", they are all evolving and have acquired changes that affect their properties. Indeed there is no one sequence for Xpr1 in mice, but many. The Xpr1 gene in most laboratory mice is altered such that the protein it makes no longer functions as a receptor for xenotropic retroviruses. In contrast, the Xpr1 gene in several strains of wild mice encodes a protein that does function as a receptor for xenotropic retroviruses, as does the human Xpr1 protein.

In addition, it would be nice if this poster included the entire section from my lab web page:

In later studies we focused on how XMRV might be involved in CFS, and found that XMRV could kill neuroblastoma cells, indicating a neurotoxic activity of XMRV that might be involved in CFS. This toxic effect was mediated by interaction of the virus with its cell-entry receptor, Xpr1, which we recently found was an atypical G-protein-coupled receptor that regulated cAMP levels in the neuroblastoma cells. XMRV infection blocked Xpr1-mediated signaling resulting in reduced cAMP levels and cell death.

However, we continue to study the role of Xpr1 in cell biology, and are looking for possible natural ligands that might regulate Xpr1 activity. Xpr1 orthologs are present in many organisms, including plants, animals and fungi, and XMRV has provided a window into what may be an important regulatory pathway in a wide range of living things

Miller/Eco

 

[ukxmrv]

Barb, all we are going to get are opinions though. That is opinions from scientists, researchers, patients.

My own method is that I don't wait to be told what to think about any paper. I examine the evidence and ask around from the people I know with a scientific, medical or other useful background. I realise that this is different and I'm not trying to say your way of reviewing each paper is wrong. It's just a different way of doing things.

During the early years of ME I told to go to medical school libraries and read papers there. I did the same thing (i.e. then asked people I knew and wrote to doctors etc before email) and this is just a method that has worked well for me.

Ecoclimber, I am grateful to you for asking Dr Miller and for posting his response here. The more we can learn the better we can understand the issues.

 

[natasa778]

I (Miller) will clarify the inaccuracy in post 28 that refers to my work. Mice do have an Xpr1 gene that is expressed in most mouse tissues. One can verify that mice have an Xpr1 gene by doing a blast search for sequences similar to the human Xpr1 cDNA, GenBank accession NM_004736.3 . A little way down the list is GenBank accession NM_011273.2 , the Mus musculus xenotropic and polytropic retrovirus receptor. But the key point is that while Xpr1 genes present in many organisms are related and are therefore all called "Xpr1", they are all evolving and have acquired changes that affect their properties. Indeed there is no one sequence for Xpr1 in mice, but many. The Xpr1 gene in most laboratory mice is altered such that the protein it makes no longer functions as a receptor for xenotropic retroviruses. In contrast, the Xpr1 gene in several strains of wild mice encodes a protein that does function as a receptor for xenotropic retroviruses, as does the human Xpr1 protein.

Thanks.

In addition, it would be nice if this poster included the entire section from my lab web page:

In later studies we focused on how XMRV might be involved in CFS, and found that XMRV could kill neuroblastoma cells, indicating a neurotoxic activity of XMRV that might be involved in CFS. This toxic effect was mediated by interaction of the virus with its cell-entry receptor, Xpr1, which we recently found was an atypical G-protein-coupled receptor that regulated cAMP levels in the neuroblastoma cells. XMRV infection blocked Xpr1-mediated signaling resulting in reduced cAMP levels and cell death.

However, we continue to study the role of Xpr1 in cell biology, and are looking for possible natural ligands that might regulate Xpr1 activity. Xpr1 orthologs are present in many organisms, including plants, animals and fungi, and XMRV has provided a window into what may be an important regulatory pathway in a wide range of living things

Miller/Eco

This is the exact section that I copied, no? But just in case you are referring to my last comment, I guess it was just the question of people already being aware of the views expressed on that web page re xmrv origin...

 

[Dusty Miller]

Thanks.



This is the exact section that I copied, no? But just in case you are referring to my last comment, I guess it was just the question of people already being aware of the views expressed on that web page re xmrv origin...

You are correct, this is what you posted. The section that was left out was:

"It is now known that XMRV was not present in the original tumor from which 22Rv1 cells were derived, but instead, arose during passage of these cells as a xenograft in mice. The original findings of XMRV association with prostate cancer and CFS have not been confirmed by others, including us, and it has become clear that detection of XMRV in early studies was due to contamination with XMRV virus from 22Rv1 cells, and later, with plasmid clones of XMRV."

(see http://sharedresources.fhcrc.org/profile/miller-dusty)

I believe this section summarizes what the majority of scientists now believe.

 

[natasa778]

Yes that is what I meant in comment above. When you say "The original findings of XMRV association with prostate cancer and CFS have not been confirmed by others," ----- the discussion here has been on the need to ditch PCR as becoming clear it is not enough, and to use NGS instead or alongside PCR ...

 

[currer]

"It is now known that XMRV was not present in the original tumor from which 22Rv1 cells were derived, but instead, arose during passage of these cells as a xenograft in mice"

From the discussion on this thread it seems to me that there are still a lot of valid questions about the origin of the "XMRV" sequence that Paprotka has left unanswered.

 

[anciendaze]

It would be interesting to know if Dr. Miller still holds the position that such a recombination event producing a human pathogen is so improbable that it could only occur in a laboratory mouse with a human xenograft. I have the suspicion that some of the big numbers thrown around in that debate were based on assumptions which now render them meaningless.

 

[currer]

Remember this paper? http://www.ncbi.nlm.nih.gov/pubmed/21750403
Zhang "Frequent detection of XMLV in human cultures established from mouse xenografts"
They recombine all the time......and the recombinants are adapted to the human cell line they are in.

 

[August59]

I just want to reiterate my appreciation of the level of knowledge, the candid points of veiw and the clear respect all posters have had for the others post and opinions. This is the epitome of what a Forum and subsequent threads and post are all about and I truly extend my thanks to every single person contributing to this thread

Phoenix Rising is second to no one!!!!

 

[Dusty Miller]

Yes that is what I meant in comment above. When you say "The original findings of XMRV association with prostate cancer and CFS have not been confirmed by others," ----- the discussion here has been on the need to ditch PCR as becoming clear it is not enough, and to use NGS instead or alongside PCR ...

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. For example, Aloia et al. (Cancer Research 70:10028, 2010) could not find XMRV in prostate cancer samples from almost 800 patients. In the case of CFS, Knox et al. (Science 333:6038, 2011) couldn't find XMRV or other MLVs in "61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive". These are just a few of the papers, many of which I'm sure you are aware of.

Regarding NGS (next generation sequencing), this will not solve the problem of detection of XMRV in those with CFS. It is actually less sensitive than PCR for detection of specific pathogens, such as XMRV. You are correct when you say it should be used alongside PCR, as it provides different information. But, NGS will not detect XMRV in patients when PCR has already failed. The reason for this is that PCR, when done properly, has the capability to detect a single copy of a particular DNA sequence in a vast excess of other DNAs. NGS can reveal the broad distribution of DNA sequences present in a sample, but doesn't have the sensitivity of PCR for detecting a sequence present at one copy in a large mixture of other DNAs.

 

[Dusty Miller]

Remember this paper? http://www.ncbi.nlm.nih.gov/pubmed/21750403
Zhang "Frequent detection of XMLV in human cultures established from mouse xenografts"
They recombine all the time......and the recombinants are adapted to the human cell line they are in.

Yes, I know this paper, but it is old news. In 1977, Suzuki et al. (Gann 68:99, 1977) reported similar findings that human cancers transplanted into mice readily acquired retroviruses (type C viruses). Here's the Abstract:

"Type-C virus particles were revealed by electron microscope in 6 of 9 tumours of cultured and biopsied human cancers heterotransplanted into nude mice. Some tumours in nude mice were explanted for in vitro cultivation. The virus particles were also found in the cultures derived from the virus-positive tumors. They were mostly found extracellularly, but the particles in budding process were also encountered frequently. Cytological study and karyotype analysis of the cultured cells proved these virus-releasing cells are thus of human origin. From the close correlation between the statistical virus counts and the complement fixation titers for murine gs antigen of the tumors and their cultures, these viruses propagated in human cancer cells were confirmed to be infectious viruses of nude mouse origin. The virus replicating in human cancer cells was readily infected in some of innocent human cancer cells by co-cultivation. It is to be emphasized that infection of animal endogenous viruses on heterotransplanted human cancer cells is a bothersome contamination for human cancer research, especially when searching for a human tumor virus candidate."

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.

Moreover, there are mice that produce high levels of a xenotropic retrovirus in their blood. The mice are called New Zealand black mice, and they carry an infectious copy of a xoentropic retrovirus in their genomic DNA. No need for recombination here! As I remember, they start of produce this xenotropic retrovirus, called the NZB retrovirus, early in life, but it is not pathogenic in these mice. This virus is not a human pathogen, even though it can easily infect human cells in culture.

But I digress. To my knowledge, there is no good evidence for the presence of XMRV in humans, let alone that it causes disease in humans, and many excellent papers that report it is not present.