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

[Jemal]

Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice using deep sequencing
Mayer J, Mazzoni CJ, Greenwood AD.

Department of Human Genetics, Center of Human and Molecular Biology, Medical Faculty, University of Saarland, 66421 Homburg, Germany.

Abstract
It has recently been reported that the xenotropic murine leukemia virus-related virus (XMRV) derives from a laboratory recombinant. However, sequences with characteristics of the 5' half of XMRV (termed PreXMRV-2) have been identified in several laboratory mouse genomes and cell lines suggesting parts of the XMRV genome exist as naturally occurring retroviruses in mice. We compare here PreXMRV-2 gag sequence diversity in mice to that of reported XMRV-like sequences by testing a panel of wild mouse and common inbred laboratory mouse strain genomic DNAs and by using high throughput amplicon sequencing. Sequences with features typical of previously reported PreXMRV-2 sequences, among them a 24 nt deletion, were repeatedly identified in different wild mice and inbred mouse strains within a high background of non-XMRV-like sequences. However, Sanger sequencing of clones from amplicons failed to retrieve such sequences effectively. Phylogenetic analysis suggests that PreXMRV-2 gag sequences from mice, cell lines and patient samples belong to the same evolutionarily young clade and that such sequences are diverse and widespread within Mus musculus domesticus and laboratory mice derived from this species. No evidence of PreXMRV-2 like gag sequences could be obtained outside of the Mus musculus lineage. The results suggest that accurate determination of presence, absence and relationships of specific murine retroviral strains benefit greatly from deep sequencing analysis.

http://www.ncbi.nlm.nih.gov/pubmed/22771940

 

[Bob]

Thanks Jemal.

 

[Ecoclimber]

http://okeefe-lab.blogspot.com/2012/07/next-gen-sequencing-reveals-that-pre.html

Next gen sequencing reveals that Pre-XMRV-2 is part of an evolutionarily young clade and more issues with PCR amplification of MLV-related sequences


There are a load of papers I'd love to blog about right now, but have been too busy writing grants to get them up -- this one however caught my eye today -- I think we have a lot of very interesting data yet to be revealed by next-gen sequencing - this paper, in Virus Research, compares pre-XMRV-2 sequences found in both wild-mice and inbred mouse populations. It also seems that despite the fact that the virus appears to be a polytropic non-ecotropic virus [thus can infect other wild mice], not all wild mice in the same geo area carry the virus (in this case, Germany), and phylogenetic analysis implies that the clade is evolutionarily young and wide-spread amongst M.m. domesticus. Also interesting is that the authors, Mayer et al., demonstrate the issues involved with PCR amplifying the gag region from these mice - and the utility of high throughput deep sequencing. If you have access to Virus Research, you can read all about it - click here for the abstract - Denise S. O'Keefe, PhD.

Eco

 

[Bob]

Thanks Eco

 

[VillageLife]

So part of the xmrv virus is a poly?

Dr Ruscetti always said they were finding what looked to be a family of gammaretroviruses.

 

[VillageLife]

Dr Ruscetti and dr mikovits have not been treated well by the scientific community.
They have been working so hard studying these mouse retroviruses, trying to help 20 million ME/CFS sufferers

 

[barbc56]

Dr Ruscetti and dr mikovits have not been treated well by the scientific community.
They have been working so hard studying these mouse retroviruses, trying to help 20 million ME/CFS sufferers

So have other scientest.

Barb C.

 

[barbc56]

Eco, what does this mean as far as ME/CFS and the contamination theory.
Thanks.
Barb C.:>)

 

[currer]

I wonder what Vincent Racaniello makes of this paper, which undermines the reasoning behind the Paprotka paper he so enthusiastically supported?

 

[barbc56]

currer, post: 280594, member: 1504" I wonder what Vincent Racaniello makes of this paper, which undermines the reasoning behind the Paprotka paper he so enthusiastically supported?

Does it? What makes you think this undermines the Paprioka paper? It very well may but I have no idea.

Any blogs/ perspectives on this study?

If theorctically this study affects the Paaprka paper, and I have no idea if it this is true or not, I would think Racaniello, would go with the present state of knowledge. If a hypothesis turns out to be false, you move to the next hypothesis. It's a process. A study that that does not support the hypothesis it's not a negative dire situation as you still learn from the experiment and the scientific community, in theory, scientist should know this. Sometimes, as a lay person, It's still wrap my head around this.

In practice I know this sometimes doesn't work. I've seen first hand, in a university "publish or perish" atmoshere, where egos are big, politics rampant, and it can get very ugly, especially when it concerns tenure. It's looked upon as an embarassment where the scientest works, but not necessarily negative in the science community.

Hopefully other scientiest would look at this research, analysize it and be more objective about it. IMHO this is what happens most of the time. I'm sure this is also true in other situations.

That's why I like Silverman. Although, honestly, most scientest would (hopefully) do the same thing.

Barb C.:>)

 

[Bob]

I might have misinterpreted some of it, as I haven't seen the full paper yet, and we need to read the full paper to fully understand it...

But my basic understanding and interpretation, based on the abstract and on O'Keefe's blog, is as follows...


PreXMRV2-like gag sequences have been detected in some wild mice.
(PreMXRV2 is a mouse virus, and shares a considerable proportion of its genome with XMRV.)

The authors seem to suggest that the preXMRV2-like sequences belong to a relatively recent virus (i.e. recently evolved or created by recombination).
It hasn't spread to all mice in the geographical area in which it has been found.

There also seems to be some discussion that gag genes, for XMRV-like viruses, cannot be reliably detected with methods other than deep sequencing, and that deep sequencing is necessary for reliable test results.

I don't fully understand what the consequences might be, except to say that if preXMRV2 is found in the wild, then preXMRV1 might also be found in the wild. In which case, it might be quite likely that an XMRV-like virus is also found in wild mice. If I remember correctly (memory not reliable), Paprotka et al didn't determine definitively how preXMRV1, preXMRV2 and XMRV got into the cell line, but made an assumption that preXMRV1 and preXMRV2 was transmitted into the cell line via the mice used in the laboratory, and that they recombined to form XMRV. I guess that if preXMRV1 and preXMRV2 are found in the wild, with the possibility that XMRV-like viruses are also found in wild mice, then that would increase the possibilities of how the cell line became infected with XMRV. It increases the chances that the cell line became infected with XMRV through external contamination. But I don't think that finding preXMRV2 in the wild actually changes anything fundamental by itself, unless they also find preXMRV1 or an XMRV-like virus.

Also, the stuff about gag genes not being reliably detectable, with methods other than deep sequencing, might have implications with regards to detecting MLV-like viruses, but I'd need to see the full paper to see the details.

I might have misunderstood, so I'd be interested in anyone else's thoughts.


Edit:

I'm not sure what they mean by the following, because I'm not aware of preXMRV2 sequences having been found in patient samples, except in the XMRV genome. Did they find preXMRV2 in patient samples in this study, or are they referring to preXMRV2 sequences in XMRV?:
"Phylogenetic analysis suggests that PreXMRV-2 gag sequences from mice, cell lines and patient samples belong to the same evolutionarily young clade and that such sequences are diverse and widespread within Mus musculus domesticus and laboratory mice derived from this species."

In any case, they seem to be saying that the sequences found in patients, and in cell lines, and in the wild mice, and in laboratory mice, are closely related with each other. I'm not sure what the implications are, except possibly to say that the XMRV detected in patient samples is related to the preXMRV2 found in wild mice and lab mice and cell lines. Unless they've detected preXMRV2 in patient samples in this study.


The abstract seems to hang partly on the final sentence, which suggests that accurate testing for XMRV-related sequences is more reliably carried out by deep sequencing:
"The results suggest that accurate determination of presence, absence and relationships of specific murine retroviral strains benefit greatly from deep sequencing analysis."

 

[currer]

This paper shows that finding pre-xmrv2 sequences in human samples cannot automatically be ascribed to "laboratory contamination" from endogenous retroviruses in one strain of laboratory mouse bercause these sequences exist in the wild as mouse viruses.

Paprotka argued that "XMRV" was lab contamination and that the characteristic sequence was unique to a laboratory mouse. If pre xmrv 2 can replicate it will be diverse and all arguments about characteristic sequences void.

The Mayer paper also suggests that deep sequencing is the only effective way to reveal the presence of these viral sequences. PCR cannot find them.

 

[alex3619]

I have never agreed the Paprotka paper was conclusive. The reasons for my not agreeing are supported by this current paper, essentially along the lines pointed out by Bob in post 11.

However, like all findings we need additional lines of evidence to be even close to sure. This is a good first step, but we will not begin to see the full implication until more studies are done.

In reply to currer in post 12, Paprotka put forward the view that pre-XMRV2 was naturally present in mice at some point - how else could it have been found in their DNA? The finding of this in the wild might also support Paprotka. The finding of pre-XMRV1 in the wild would strengthen that line of support. However, at the same time it would undermine the argument that the two combined in the lab due to it being statistically verging on impossible for it to occur in an isolated instance more than once.

The existance of these pre-XMRVs in the wild, or even of XMRV itself, will not disprove Paprotka. They will just create doubt.

To refresh the memories of those who were not aware, my argument is that as the number of potential viral sources increased, the probability of XMRV arising in the wild would increase. There are a lot of rodents in the world, not to mention other mammalian vectors. So many that the likelihood of XMRV arising in the wild, many many times, is very high. This might mean it is more likely that XMRV contaminated the tissue culture rather than arising in it, especially now that we know that XMRV can easily pass through standard precautions to infect cultures.

Bye, Alex

 

[currer]

Paprotkas argument was that both pre xmrv 1 and 2 were ERVs that could only recombine to create "xmrv" in the laboratory by artificial means (due to human intervention and a one-offf event)
"We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10(-12)); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event." (Paprotka)

This Mayer paper casts doubt on that. It suggests that pre xmrv 2 could be an infectious retrovirus in wild mice and therefore capable of replicating in the wild.
So the pre xmrv 2 gag segment could be part of an infectious retrovirus that just needs complete sequencing.....not "XMRV", but HGRV perhaps......look how many gag sequences have been found by Mikovits inher samples.

 

[Bob]

To refresh the memories of those who were not aware, my argument is that as the number of potential viral sources increased, the probability of XMRV arising in the wild would increase. There are a lot of rodents in the world, not to mention other mammalian vectors. So many that the likelihood of XMRV arising in the wild, many many times, is very high. This might mean it is more likely that XMRV contaminated the tissue culture rather than arising in it, especially now that we know that XMRV can easily pass through standard precautions to infect cultures.

Yes, it seems so much more likely that an XMRV-like virus would have been created in the wild, rather than in the lab, because there would be almost unlimited opportunities for it to be created. Whereas, there was a very limited opportunity for XMRV to be created in the single cell line.
Although, the argument in favour of it being created in the cell line, is that XMRV has an affinity for human tissue, so it would have to have either been created in the cell line, or to have entered the cell line via contamination from lab workers infected with XMRV.

 

[Bob]

This paper shows that finding pre-xmrv2 sequences in human samples cannot automatically be ascribed to "laboratory contamination" from endogenous retroviruses in one strain of laboratory mouse because these sequences exist in the wild as mouse viruses.

So if we assume that some of the positive XMRV findings in humans could actually have been preXMRV2-like viruses, and not XMRV, then that widens the field and widens the possibilities?

By "preXMRV2 sequences in humans samples", you mean previous XMRV research, right?

Paprotka argued that "XMRV" was lab contamination and that the characteristic sequence was unique to a laboratory mouse. If pre xmrv 2 can replicate it will be diverse and all arguments about characteristic sequences void.

I can't work out what you mean there currer. What are you referring to in relation to arguments about character sequences being 'void'?

i.e. What would the implications be, for a diverse genome in preXMRV2-related viruses, in relation to the Paprotka study?

Paprotkas argument was that both pre xmrv 1 and 2 were ERVs that could only recombine to create "xmrv" in the laboratory by artificial means (due to human intervention and a one-offf event)
"We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10(-12)); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event." (Paprotka)

This Mayer paper casts doubt on that. It suggests that pre xmrv 2 could be an infectious retrovirus in wild mice and therefore capable of replicating in the wild.
So the pre xmrv 2 gag segment could be part of an infectious retrovirus that just needs complete sequencing.....not "XMRV", but HGRV perhaps......look how many gag sequences have been found by Mikovits inher samples.

I'm getting a bit confused now!

What preXMRV2 gag segment are you referring to, where you say it needs complete sequencing? Are you referring to Mikovits' research?

If preXMRV2 can jump ship, so to speak, and infect a mouse which has preXMRV1, then the two could recombine in the wild mice or in lab mice, even if preXMRV1 is non-infectious.

And the new research suggests that there are more opportunities for XMRV-like viruses to be created in the wild, rather than just in the lab.

But it doesn't answer the specific question about XMRV 22rv1, which has an affinity for human tissue, as that couldn't have been created in mice or mouse tissue. However, a mouse virus, similar to XMRV, could have been created in mice, which could then potentially have jumped to humans to create XMRV.

I suppose that there are endless other possibilities as well, as so little full sequencing has been carried out so far, and so little deep sequencing.

And we need full sequencing of viral genomes (with latest technology), and not just sequencing of gag or pol or env sequences.

 

[currer]

Hi I modified my earlier post, Bob so my quote is not accurate! That will teach me to post without thinking!

 

[Bob]

Have I've misunderstood the Paprotka paper (very possible), in that the gag gene for XMRV is proposed to have come from preXMRV1, and not preXMRV2 (see the diagrams on the last two pages of the Paprotka paper on the PDF version). So I'm not sure how this new study fits in with XMRV, as it was only studying gag sequences.

So it also makes me re-ask the question about this study, about comparing preXMRV2 sequences from 'patient samples'. What preXMRV2 gag sequences in patient samples are they referring to?

But if preXMRV2 has been found in the wild, I think it makes it very likely that preXMRV1 will also be found in the wild.

 

[Bob]

Hi I modified my earlier post, Bob so my quote is not accurate! That will teach me to post without thinking!

I've edited my earlier reply to you currer.

 

[Mula]

It is prexmrv2 which shares a gag with xmrv.