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

[natasa778]

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

Yes and the logic tells us that what comes up must come down To have a pathogen, or a group of pathogens that are sooo fantastically capable of infecting (with ease and grace most human cells/tissue they come near, that are known to cause the type of neuroimmune disease that are still of mysterious etiology in humans, and as you said cancers, that love to throw wild recombining parties with other rv's and host erv's, and finally that are known to go into long latency periods during which they are absent from blood and can only be detected hiding deep in some organs, the logic tells us that we should throw a wider and deeper net on this one.

 

[currer]

"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."

The point I am making is not that the gene vectors themselves are dangerous, as they can be modified.
I am drawing your attentuion to the danger of recombination between such a gene vector and wild MLVs IF THEY ARE PRESENT in the human population. - Now I accept that this is not proven, and may be unlikely. But I am concerned that commercial pressures may overrride the need to look for these retroviruses in humans or disregard sich signs if they appear.

This is the concern of the Dong paper http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003144

That these are genuine POSSIBILITIES is shown by the concern of the authors.

"The fact that a popular class of retroviral vector could readily become infectious in the presence of natural human virus has to be seriously considered whenever these constructs are being used, especially in a clinical setting. The most obvious concern is the possibility that a vector could accidentally encounter XMRV and would spread in human populations. In the case of gene therapy, the therapeutic construct might spread from the intended tissue target throughout the organism or even to additional individuals. An even more deleterious, albeit less probable, event might be an accidental escape of an experimental vector that harbors an oncogenic “cargo”. Modern MLV-based vectors have undergone extensive engineering to improve their performance in specialized tasks [1]. For example, mutations in the long terminal repeats have been used to improve expression, especially in embryonic cells[17]. An escape of such constructs would open a possibility for recombination with natural viruses, potentially increasing pathogenicity of the latter."

Now this all hinges on whether such MLVs are really present as infections in the human population. This is why posters on these forums have expressed such concern at the seeming eagerness to dismiss and use disreputable means to discredit those scientists who raise such concerns.

We are not averse to a fair and open debate. The question is - Whether it IS fair and open?

 

[barbc56]

Yes and the logic tells us that what comes up must come down To have a pathogen, or a group of pathogens that are sooo fantastically capable of infecting (with ease and grace most human cells/tissue they come near, that are known to cause the type of neuroimmune disease that are still of mysterious etiology in humans, and as you said cancers, that love to throw wild recombining parties with other rv's and host erv's, and finally that are known to go into long latency periods during which they are absent from blood and can only be detected hiding deep in some organs, the logic tells us that we should throw a wider and deeper net on this one.

We are talking Science here and not speculation.

I may be wrong but I think casting a wider net also increase the chances of picking up background noise, bits of viruses, contamination.

I wouldn't be surprised if science knows how to differentiate between this and a true RV that infects and also causes disease. XMRV, whatever we are calling it now, is infectious but you can't draw from that the conclusion that it causes illness.

As far as taking precautions I would think that woud be best standard practice.

Barb C.:>)

 

[barbc56]

Now this all hinges on whether such MLVs are really present as infections in the human population. This is why posters on these forums have expressed such concern at the seeming eagerness to dismiss and use disreputable means to discredit those scientists who raise such concerns.

We are not averse to a fair and open debate. The question is- Whether is IS fair and open?

Who is "we:? I don't feel that scientist are eagar to dismiss and use disreputable means, etc., etc. etc. Using conspiracy theories scraps the bottom of the barrel as far as logic goes.

Debate should be free and open and that means respecting other's opinions, debate the facts and not use personal slurs
.
IMHO, I still don't understand why people are holding on to the fact that XMRV is responsible for our illness when the science is showing otherwise. If the science was showing XMRV is indeed causing illness, I would go with that.

We all have a right to our opinions but also a responsibility express those opinions in a respectful way. I will respect your opinion but it doesn't mean I have to believe it.

Just saying.

Barb C.:>)

 

[Firestormm]

Christ I'm no scientist Barb. Flippin' heck. Nah I've always taken care to declare my layman's status. This has been a very interesting thread. All comments have been very good to read. Lots of care taken. I just feel Currer stepped into the realm of subjective interpretation a wee bit and a couple of other err... outbursts might dissuade a contribution from a genuine scientist willing to engage is all. Anyway, it's all about Mikovits again today it would seem.

 

[Marco]

Who is "we:? I don't feel that scientist are eagar to dismiss and use disreputable means, etc., etc. etc. Using conspiracy theories scraps the bottom of the barrel as far as logic goes.

Debate should be free and open and that means respecting other's opinions, debate the facts and not use personal slurs

We all have a right to our opinions but also a responsibility express those opinions in a respectful way. I will respect your opinion but it doesn't mean I have to believe it.

Just saying.

Barb C.:>)

Barb. I'm very much on the sidelines of this debate and am grateful to Dr Millar for sparing the time to engage here and respond to some fairly technical points.

 

[jace]

Dr. Miller said

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?

How long is it now, that they have had to correctly annotate the Genbank entry? Two years, or three?
They have an env protein, but a protein can be identical for many different viruses. To get the sequence from that they must have extroplated backwards, which you cannot do accurately.

 

[Dusty Miller]

Yes and the logic tells us that what comes up must come down To have a pathogen, or a group of pathogens that are sooo fantastically capable of infecting (with ease and grace most human cells/tissue they come near, that are known to cause the type of neuroimmune disease that are still of mysterious etiology in humans, and as you said cancers, that love to throw wild recombining parties with other rv's and host erv's, and finally that are known to go into long latency periods during which they are absent from blood and can only be detected hiding deep in some organs, the logic tells us that we should throw a wider and deeper net on this one.

Nicely stated.

However, I will bring up some other data that I remember from my early days in retrovirology. Not totally sure if all of it is correct. There are many murine leukemia viruses that can infect laboratory mice, unlike the xenotropic viruses that we have been discussing. They replicate in mouse cells just as well if not better than XMRV replicates in human cells. I'll use Moloney murine leukemia virus (MoMLV) as an example. So what happens if you inject MoMLV at a high dose into adult mice? The virus replicates, and can be found in mouse blood for less than a week, until the mouse immune system takes it out. As far as I know, the mice are completely healthy after that. So, how does MoMLV cause leukemia? One needs to inject the virus into newborn mice (within two days of birth) when the mouse immune system is not yet fully developed. Then you get persistent virus replication, and eventually leukemia, due to virus integration near and activation of mouse cellular oncogenes. So, it is somewhat difficult to cause disease in mice with this mouse virus that is structurally similar to XMRV.

I still agree with you that we need to be careful with these viruses, and leave open the possibility that might be involved in human disease.

 

[natasa778]

XMRV, whatever we are calling it now, is infectious but you can't draw from that the conclusion that it causes illness.

LOL neither can you draw the conclusion that it doesn't. Better safe then sorry etc.

As far as taking precautions I would think that woud be best standard practice.

Yes you probably would. I wouldn't.

 

[currer]

I still agree with you that we need to be careful with these viruses, and leave open the possibility that might be involved in human disease.

Thank you for saying this Dr Miller. I hope research continues whatever the conclusions of the Lipkin study on the "XMRV" sequence.

 

[JT1024]

Below is evidence that the FDA is taking retroviruses seriously - not only due to contaminated research cell lines but also being present in biologicals made from mammalian cells. Excerpts are below but I've attached the actual 3 day program.

PDA/FDA Adventitious Agents and Novel Cell Substrates: Emerging Technologies and New Challenges

November 2-4, 2011
Hilton Hotel | Rockville, Maryland
Program Agenda

Wednesday, November 2, 2011

Welcome and Opening Remarks
Arifa Khan, PhD, Senior Investigator, CBER, FDA

Plenary Session 1: Opening Keynote Presentation
Moderator: Arifa Khan, PhD, Senior Investigator, CBER, FDA

Based on past experience, this talk will present potential safety concerns associated with the use of biological raw materials, including cell substrates, and also focus on risk assessment for adventitious agents in biologicals.

Plenary Session 2: Adventitious Agent Testing and Emerging Methods Part I

This session will begin with an overview of the current assays that are used for virus detection with an emphasis on their benefits and limitations, and describe how those limitations have generated a need for new technologies. This will be followed by a broad talk on one of the new technologies, Next-Gen sequencing, in which the principles behind the technology and the various platforms available will be described.

Risks Associated with Retroelements: Lessons from Mammalian Systems
Jonathan Stoye, PhD, Head of the Division of Virology, MRC National Institute for Medical Research

Plenary Session 12: Adventitious Agents and Raw Materials Part I
Moderators: Zenobia Taraporewala, PhD, CMC Reviewer, CBER, FDA and Mark Plavsic, PhD, Senior Director and Corporate
Biosafety Advisor, Genzyme a Sanofi Company

As long as therapeutics and vaccines are produced in biological systems, there is the risk of product contamination by adventitious agents, mainly through the use of raw materials of animal or non-animal origin in the manufacturing processes. Manufacturers control for this risk by careful raw material selection, vendor qualification, raw material and cell bank testing, and raw material risk assessments. However, recent incidents with the detection of adventitious agents in commercial products have highlighted the practical hurdles manufacturers face with the detection and identification of adventitious agents in raw materials due to low level contamination, lot-to-lot variation, limited sampling, assay sensitivity, and sample ‘matrix’ effect. This session will discuss safety issues associated with the use of animal and certain non-animal derived raw materials, and ways to mitigate the risk of contaminationby adventitious agents. In the session, speakers will address challenges in adventitious agent screening/testing and reduction strategies for raw materials used in banking and batch production

 

[natasa778]

So what happens if you inject MoMLV at a high dose into adult mice? The virus replicates, and can be found in mouse blood for less than a week, until the mouse immune system takes it out. As far as I know, the mice are completely healthy after that. So, how does MoMLV cause leukemia? One needs to inject the virus into newborn mice (within two days of birth) when the mouse immune system is not yet fully developed. Then you get persistent virus replication, and eventually leukemia, due to virus integration near and activation of mouse cellular oncogenes. So, it is somewhat difficult to cause disease in mice with this mouse virus that is structurally similar to XMRV.

Nice example: so what about a scenario where after a week, just when we think that 'the mouse immune system takes it out', the virus actually goes into deep latency, somewhere safe and away from the immune system (say CNS), only to come out again during pregnancy/postpartum/nursing? Has anyone ruled this out? Has anyone looked hard and deep into animal tissue after alleged clearing events? What about monkey XMRV study, which found the same thing? Has anyone ever followed these infected but cleared animals for a REALLY long period of time, say equivalent of human 20, 30 years, then exposed them to immune stressors, pregnancies etc, and then looked for presence of those viruses and whether the animals are still completely healthy.

Also has anyone looked at what happens to offspring of those animals who have 'cleared the retrovirus' from blood in the long run, what happens during pregnancy/postpartum/nursing, is it possible that those retroviruses come out of latency then in order to infect the offspring? Has anyone ruled that out? Has anyone tried exposing either the pregnant animals or their offspring to immune stressors etc things that are known to reactivate retroviruses? What about once the offspring reach sexual maturity?

I just think that if we really want to explore possibilities that retroviruses might be involved in human disease we need to think laterally and not be worried about what we might uncover.

 

[currer]

(from the FDA programme)
Technical Challenges and Considerations for Applying Next-generation Sequencing to Detection of Adventitious Agents
Christopher Wang

Isnt PCR reliable enough?

 

[Dusty Miller]

(from the FDA programme)
Technical Challenges and Considerations for Applying Next-generation Sequencing to Detection of Adventitious Agents
Christopher Wang

Isnt PCR reliable enough?

PCR is only useful if you know what you are looking for. To perform PCR, short DNA 'primers' are made that bind to regions of the DNA of interest that are fairly close together, and thermocycling with DNA polymerase is performed to amplify this specific sequence. If you don't know the sequence of the virus you are trying to find, you are often out of luck with PCR. Next generation or 'deep' sequencing provides sequences for all of the DNA present in a sample without requiring any knowledge of what sequences might be present. This is useful to detect unknown viruses that might be present in cultured cells, or in blood cells of CFS subjects for example. However, there are still problems the sensitivity of this technique, and with sifting through the large amount of data generated by deep sequencing to find some new potential pathogen. For example, when sequencing a human DNA sample that might contain DNA from a new pathogen, one must get rid of all the sequences arising from the billions of bases that comprise the human genome. These are just some of the technical issues associated with deep sequencing.

 

[Dusty Miller]

Dr. Miller said

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?​

How long is it now, that they have had to correctly annotate the Genbank entry? Two years, or three?
They have an env protein, but a protein can be identical for many different viruses. To get the sequence from that they must have extroplated backwards, which you cannot do accurately.

You may have gotten your wish. When I look at the current 22Rv1/CWR-R1 sequence in GenBank, http://www.ncbi.nlm.nih.gov/nucleot...ank&log$=nucltop&blast_rank=1&RID=0V2U9EKY01S , I see the following annotation:

mat_peptide 5754..7691
/product="putative envelope polyprotein"

By the way, it's call 'putative' because no one has yet isolated and determined the amino acid sequence of the viral Env protein. But we all believe the Central Dogma: DNA -> RNA -> protein ... at least most of the time.

 

[JT1024]

Technologies have failed to detect novel adventitious agents so there are many efforts underway to remedy the situation.

http://www.slideshare.net/mewiebe/pda-annual-mtg-2012-caacb-talk
http://cbi.mit.edu/research-overview/caacb/

The MIT Consortium on Adventitious Agent Contamination in Biomanufacturing
Safe and Reliable Manufacture of Biotherapeutics

One challenge in the safe and reliable production of biopharmaceuticals is mitigating the risk of cell culture contamination by adventitious agents such as viruses or mycoplasma. Currently, industrial knowledge about contaminating adventitious agents and successful approaches to combat these specific agents is largely limited to each company’s individual experience. As such, a central database of accumulated industry knowledge and experience does not exist. The collection, analysis and dissemination of the full body of industry experience would enhance the reliable manufacture and supply of such safe and effective medications.

Purpose and Outcome of the CAACB

The MIT CBI, along with several founding member companies, has launched the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB). The goals of the consortium are: the confidential collection and analysis of industry adventitious agent contamination data; and the dissemination of the most effective industry practices used to combat contaminations during the reliable manufacture of life-saving biotherapeutic medications. MIT is an ideal venue to host this initiative due to MIT’s existing track record with industrial collaborations and its strong faculty experience in solving complex systems engineering problems.
A comprehensive analysis of adventitious agent contamination data will be highly valuable as an industry “lessons learned” exercise. Such information should inform the industry on the best practices to mitigate the risks that lead to adventitious agent contamination. Specifically, the CAACB seeks to develop a comprehensive understanding of adventitious agents encountered, the source of such agents, and a risk-based analysis of the most effective barriers to contamination. The collection, analysis and dissemination of this information should enhance the uninterrupted supply of vital, safe and effective biotherapeutic medications. This is a goal shared by both the biopharmaceutical manufacturing industry and the CAACB as we strive to combat disease.



http://www.ibclifesciences.com/ViralSafety/agenda.xml

Methods for the Identification and Reduction of Adventitious Agent Risks in Raw Materials for Live Virus Vaccine Manufacturing

Adventitious agents in vaccine and biological products have been an area of concern to regulatory agencies, manufacturers, and public health officials since the issue first arose in the early 1900s. While vaccines and the raw materials used during vaccine production are manufactured and tested in compliance with current regulations; quality control tests are broad, non-specific, and may not be capable of detecting novel or emerging adventitious agents. The possibility of adventitious agent contaminations of licensed products has recently been brought to light through the use of new analytical technologies. Such non-biased, highly sensitive technologies have been able to detect adventitious agent contaminations where conventional methods have failed to detect them. In response to these events, vaccine manufacturers have had to re-evaluate adventitious agent risks in their manufacturing processes. Legacy live virus vaccine processes are dependent on animal derived raw materials; removing animal derived raw materials from these processes, if possible, can take >10 years to realize. As such, Merck & Co. has developed a systematic approach to evaluate adventitious agent risks associated with the use of animal derived raw materials in live virus vaccine manufacturing. This approach includes the combination of FMEA and the development of a novel raw material screening program to identify adventitious agent risks in animal derived raw materials. Potential strategies for remediating the identified adventitious agent risks are also presented.

Tara Tagmyer, Ph.D., Process Scientist, Vaccine Manufacturing Sciences & Commercialization, Merck & Co.

 

[Dusty Miller]

Dusty Miller said: ↑

So what happens if you inject MoMLV at a high dose into adult mice? The virus replicates, and can be found in mouse blood for less than a week, until the mouse immune system takes it out. As far as I know, the mice are completely healthy after that. So, how does MoMLV cause leukemia? One needs to inject the virus into newborn mice (within two days of birth) when the mouse immune system is not yet fully developed. Then you get persistent virus replication, and eventually leukemia, due to virus integration near and activation of mouse cellular oncogenes. So, it is somewhat difficult to cause disease in mice with this mouse virus that is structurally similar to XMRV.​

Nice example: so what about a scenario where after a week, just when we think that 'the mouse immune system takes it out', the virus actually goes into deep latency, somewhere safe and away from the immune system (say CNS), only to come out again during pregnancy/postpartum/nursing? Has anyone ruled this out? Has anyone looked hard and deep into animal tissue after alleged clearing events? What about monkey XMRV study, which found the same thing? Has anyone ever followed these infected but cleared animals for a REALLY long period of time, say equivalent of human 20, 30 years, then exposed them to immune stressors, pregnancies etc, and then looked for presence of those viruses and whether the animals are still completely healthy.

Also has anyone looked at what happens to offspring of those animals who have 'cleared the retrovirus' from blood in the long run, what happens during pregnancy/postpartum/nursing, is it possible that those retroviruses come out of latency then in order to infect the offspring? Has anyone ruled that out? Has anyone tried exposing either the pregnant animals or their offspring to immune stressors etc things that are known to reactivate retroviruses? What about once the offspring reach sexual maturity?

I just think that if we really want to explore possibilities that retroviruses might be involved in human disease we need to think laterally and not be worried about what we might uncover.

Interesting questions. My understanding is that these simple retroviruses have no easy way to go latent. Unlike herpesviruses, for example, which have complex genetic machinery to regulate their active versus latent states, simple retroviruses like XMRV and MoMLV are always 'on'. They have strong enhancers and promoters of transcription that appear to be designed for maximum gene expression under all circumstances.

However, it is true that retrovirus expression can be suppressed by the host cell after integration of the virus, and this might be considered a form of latency. I don't know whether experiments have been done to look at the fate of such integrated but inactive viruses in animals. Certainly, it would be difficult to obtain funding to explore these issues in primates, our closest relatives, for 30 years under many different conditions, given the fact that there is little evidence that these viruses are present or cause disease in humans. In contrast, a huge and well-funded effort to understand HIV, a known human pathogen, is underway. Obviously, researchers and funding agencies must make choices about where to focus their efforts for maximum benefit.

 

[natasa778]

Interesting questions. My understanding is that these simple retroviruses have no easy way to go latent. Unlike herpesviruses, for example, which have complex genetic machinery to regulate their active versus latent states, simple retroviruses like XMRV and MoMLV are always 'on'. They have strong enhancers and promoters of transcription that appear to be designed for maximum gene expression under all circumstances.

Thanks for that. But how does that fit with what Onlamoon's study found?

Proviral DNA was found in peripheral blood mononuclear cells (PBMC) of all three monkeys for 3-4 weeks, indicating successful infection. At one month post-infection proviral DNA was no longer detected. Plasma virus was again detected in one of the positive animals on day 291, 16 days after being immunized with a mixture of XMRV proteins. This means that viral DNA had been present in this animal but was not detected. XMRV was detected in CD4+ and CD8+ T cells and NK cells, but not in B cells or monocytes.

Rhesus macaques infected with XMRV did not display obvious clinical symptoms. Analysis of peripheral blood revealed increases in the number of circulating B and NK cells. Anti-viral antibody titers were detected after infection and re-infection of animals but soon decreased.

.... The authors suggest that in Rhesus macaques, XMRV causes first an acute infection, followed by a persistent chronic infection

...

• The presence of XMRV in reproductive tract tissues is consistent with sexual transmission of infection
• After the acute phase, virus levels are very low, but there could be a different outcome in individuals with immune dysfunction
• One animal produced virus after immunization; perhaps immune activation results in cycles of virus production
• The virus has an initial acute phase followed by reactivation. The authors comment: “While our study has not linked XMRV infection with pathogenic mechanisms that might lead to prostate cancer or chronic fatigue syndrome, we submit that such link, assuming it exists, would be a temporally distant one.”
• It would be informative to determine if XMRV is present in some of the same tissues in humans that were observed to be infected in rhesus macaques

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

 

[Dusty Miller]

Thanks for that. But how does that fit with what Onlamoon's study found?



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

Good call, I stand corrected. I hadn't looked at the Onlamoon study for some time, so I read it again. It's a very dense paper, but I agree, the data indicate persistence of XMRV in rhesus macaques beyond the chronic phase of virus replication. Frankly, I can't follow all of the presentation without more effort. One caveat - these monkeys received large doses of virus (~3 x 10^6 infectious virus particles), and results following exposure to lower doses of virus, such as might be received under 'natural' conditions (biting, scratching, and during sexual intercourse), might be quite different. Interestingly, the authors state that "None of the infected animals showed any obvious clinical symptoms", indicating that XMRV would not be classified as a pathogen, at least not an acute pathogen, in these monkeys.

 

[natasa778]

Yes, but we have to consider that the animals were sacrificed shortly after, so we have no way of knowing what pathology if any the virus would cause long term... Judging by what we know about human pathogenic retrovirus HTLV (and even HIV!) the pathology arises, IF it does, after decades of asymptomatic infection, so it would not be surprising to find another slow acting virus behave in a similar manner. Actually it would be expected?