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When Science Journals are Scarier than Science Fiction

XMRV

By Kent Heckenlively, Esq.

My choice for the scariest reading of the year was recently published  in the journal Cancer Biology and Therapy and has the unwieldly title of Frequent Detection of Infectious Xenotropic Leukemia Virus (XMLV) in Human Cultures Established from Mouse Xenografts.

For those of you who may be confused by the idea of a "xenograft" I'll provide you with the definition given by the U. S. Public Health Service.  "Any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a non-human animal source or (b) human body fluids, cells, tissues or organs that have had ex vivio contact with live non-human animal cells, tissues, or organs."  This covers vaccines as well as other surgical procedures in which human tissue is manipulated prior to transplantation.

In the scientific community mouse xenografting is often used to manipulate cancer cells for research purposes, among other things.  With research that has linked XMRV (which is a xenotropic murine leukemia virus) to prostate cancer, chronic fatigue syndrome/ME, and to a lesser extent autism, scientists from Johns Hopkins University and the University of Texas Southwestern Medical Center as well as a few other institutions thought it made sense to investigate the frequency of XMLVs in human cell lines "established from mouse xenografts and to search for the evidence of horizontal spread to other cell lines."

In layman's terms the question they were asking was, "when we do xenografting with mouse biological products how often do we get XMLVs popping up in our samples?" 

The answer they found is that six out of twenty three (26%) mouse DNA free xenografts "were strongly positive for MLV and their sequences had greater than 99% homology to known MLV strains."  These samples were obtained from seven independent laboratories. 

Further on the authors wrote, "Of the 78 non-xenograft derived cell lines maintained in the xenograft culture containing facilities, 13 (17%) were positive for MLV, including XMRV, a virus strain first identified in human tissues."  (My daughter with autism has tested positive for XMRV.)  In scientific terms this is an absolute train wreck.

This means that every surgical patient receiving any biological product which used mouse tissue in any way has a one in four chance of being exposed to an XMLV.  And that also means that any biological sample which is maintained in a facility containing xenograft cultures has a 17% chance of becoming infected.

On the question of vaccines, let's just say that only 10% of the nearly 40 vaccines children are expected to receive prior to the age of five contain mouse biological products.  This translates into roughly a 100% chance that our current generation of children will be exposed to an XMLV through a vaccination.

Are you scared yet?

It gets worse.

From the article is the following passage.  "In one case XMRV virus infection to a non-xenograft colorectal carcinoma cell line RKO was demonstrated from an XMRV containing prostate xenograft derived cell line 221 Rv1 even though the two cell lines had been maintained in the same culture facility for only a few days."

The translation for a non-medical person is this:  XMRV is highly infectious and spreads easily.

How about this for scary?  "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." 

For those of you keeping score at home, the CpG islands are responsible for methylation.  Is any of this starting to sound familiar?  And the transcription start sites of genes?  Pretty damned important.

I know there are those who question the role of viruses in conditions like autism and state correctly that a well-functioning immune system will deal with any such pathogens.  I agree.  The problem is that we don't know the immune status of people in the population.   For example, the discovery of XMRV itself was preceded by the finding that men with the most aggressive forms of prostate cancer had a deficiency in their RNasel gene, lowering the amount of an anti-viral defense enzyme their body produced.  How widely is that RNasel mutation spread throughout the population?  Do you know the RNasel status of your genes?  Of your children?  And that's just what we know about.

And can toxic chemicals and heavy metals interfere with your body's immune system response to pathogens, regardless of your genetic make-up?  You betcha.

The closest example I can make is that of what happened to the native population in the Americas when Europeans crossed the ocean.  A natural barrier was breached and the native population had no immunity to our pathogens.  Some experts speculate that the diesases Europeans brough to the New World killed 90% of the native population.

While we have shared the Earth with mice and other creatures from the dawn of history, we haven't been culturing their cells, then injecting those cells into our bloodstream.  We have breached a natural barrier and we need to ask the question of what consequences have been wrought from this decision.

In the conclusion the authors write, "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.  Laboratories working with xenograft cultures should have full knowledge and understanding of the potential biological and biohazardous risks and should not distribute or publish their findings without full disclosure of the virus status of their xenograft-derived materials."

Translation for the ordinary person:  Hey, we might be infecting our lab workers!  Let's find out what diseases these viruses might cause!  And maybe much of our research has been compromised!

I've heard some commentators lament what it will take for the medical authorities to deal with this issue seriously.  They've noted with the despair the finding of XMRV in men with prostate cancer, those with chronic fatigue syndrome/ME and children with autism and how it has failed to provoke action.  They've said to me, "These people have no problem screwing over the old men, those with CFS/ME, and even the children."

With this recent finding regarding the dangers of XMLV and XMRV transmission in labs, the question becomes, "Will these same medical authorities screw the very people who work for them?"

Addition to original article - I understand a Dr. Dusty Miller from the Fred Hutchison Cancer Center has raised some questions as to my suggestion that mouse tissue which harbors XMRV may possibly end up in a vaccine.  For Dr. Miller I submit the following article:

Biologicals. 2010 May;38(3):371-6. Epub 2010 Apr 8.
Endogenous retroviruses as potential hazards for vaccines.
Miyazawa T.

Laboratory of Signal Transduction, Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan. takavet@gmail.com

Abstract
Retroviruses are classified as exogenous or endogenous according to their mode of transmission. Generally, endogenous retroviruses (ERVs) are not pathogenic in their original hosts; however, some ERVs induce diseases. In humans, a novel gammaretrovirus was discovered in patients with prostate cancer or chronic fatigue syndrome. This virus was closely related to xenotropic murine leukemia virus (X-MLV) and designated as xenotropic murine leukemia virus-related virus (XMRV). The origin and transmission route of XMRV are still unknown at present; however, XMRV may be derived from ERVs of rodents because X-MLVs are ERVs of inbred and wild mice. Many live attenuated vaccines for animals are manufactured by using cell lines from animals, which are known to produce infectious ERVs; however, the risks of infection by ERVs from xenospecies through vaccination have been ignored. This brief review gives an overview of ERVs in cats, the potential risks of ERV infection by vaccination, the biological characteristics of RD-114 virus (a feline ERV), which possibly contaminates vaccines for companion animals, and the methods for detection of infectious RD-114 virus. 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

PMID: 20378372 [PubMed - in process]

Dr. Miller also might want to read this section of the article "Of Mice and Men: On the Origin of XMRV" from the journal Frontiers in Virology and published in January of 2011:  You can read the complete text http://www.frontiersin.org/virology/10.3389/fmicb.2010.00147/full

Vaccines, Viruses, and Contamination

One of the most widely distributed biological products that frequently involved mice or mouse tissue, at least up to recent years, are vaccines, especially vaccines against viruses. Some, for instance vaccines against rabies virus (Plotkin and Wiktor, 1978), yellow fever (YF) virus (Frierson, 2010), and Japanese encephalitis (JE) virus (Inactivated Japanese Encephalitis Virus Vaccine, 1993), consisted of viruses that were cultured on mouse brains. Such vaccines were in use from 1931 (YF vaccine) until now (JE vaccine, licensed in Japan since 1954). For rabies virus, early vaccines were mainly of goat or sheep nerve tissue origin. In addition, suckling mouse brain-derived rabies virus vaccines were used in South America and France (Plotkin and Wiktor, 1978). No mouse-derived rabies vaccine was ever licensed in the USA (Dennehy, 2001). Live-attenuated YF vaccines were originally also grown on mouse brain, but an YF vaccine grown on chicken eggs (named 17D) became available in 1937, and was since the vaccine of choice in the America’s. In 1962, contamination of the 17D vaccine with oncogenic avian leukosis virus was detected both in England and in the USA, but fortunately no excess of cancer incidence among vaccines was reported (Frierson, 2010). In France, the mouse brain-derived YF vaccine was discontinued as late as 1982.

Although being the most effective means to prevent infectious diseases and to safe lives, serious contamination problems involving vaccines have occurred (Pastoret, 2010). Contamination with unrelated viruses such as the presence of hepatitis B virus (HBV) in YF vaccine preparations stemming from the use of human serum for stabilization, and simian virus 40 (SV40) and foamy viruses through the use of monkey cell cultures (Pastoret, 2010). Some vaccine viruses are inactivated before use, hopefully also inactivating any contaminating virus particles, but the contaminating virus may be more stable than the vaccine virus. For instance, SV40 is highly resistant to inactivation (Murray, 1964). Endogenous retroviruses constitute a distinct class of contaminating viruses, as these viruses are encoded by all cells of a certain species, and therefore cannot be avoided even through rigorous screening (Miyazawa, 2010). Contamination with endogenous avian leukosis viruses is a major problem for vaccine viruses grown in chicken embryos or chicken embryonic fibroblasts (Hussain et al., 2003). Infectious cat endogenous RD-114 virus has been found in several veterinary vaccines produced in cat cell cultures (Miyazawa et al., 2010; Yoshikawa et al., 2010).

Dr. Miller, I await your reply.

 Kent Heckenlively is a Contributing Editor to Age of Autism

 

Comments

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Thanks for all you do Kent.

Dear Dr. Miller:

I was gratified to receive your response, especially the first paragraph in which you said, "I appreciate your effort to understand the disease that affects your family, and to reach out to scientists who might have useful information. That said, I know very little about autism, including possible genetic or environmental components."

You and I have probably both reached the age where we're well aware of our strengths and weaknesses, and try to highlight the former, while minimizing the later. I take no offense and look upon it only as an opportunity to further connections between people who are interested in solving the medical problems which affect so many.

The problem with the little you know of autism is shared by so many. Considering that something like 50 kids came down with the measles in my state last year and it was treated as a public health emergency while at the same time approximately 5,000 were diagnosed with autism makes many of us wonder about the priorities of the public health system. Our community would feel so much better if upon a diagnosis of autism, a public health swat team swung into action giving every imaginable test to try and figure out what had gone wrong with that specific child.

You might be interested in this article I wrote in which the government confirms they are testing approximately 100 children with autism for the presence of the XMRV retrovirus. http://www.ageofautism.com/2011/06/government-confirms-testing-of-children-with-autism-for-presence-of-xmrv-retrovirus.html

I understand your frustration with the small amount of research funding presently going to XMRV research and offer an opportunity. Starting with the autism community, but branching out to other disease communities who feel they've been long neglected, including ME/CFS, Gulf War Syndrome, lyme disease, we are forming a new political party to advocate for more research dollars for these diseases.

It is called "The Canary Party" and you can access it here. http://www.canaryparty.org/ I would consider it an honor if you joined us.

All the best,
Kent Heckenlively

Dr. Miller:
I appreiate you response, thank you for apologizing, there have been so many times I am talked to in such a demoralizing way, and not believed in what I witnessed, it is very frustrating and many here are very sensitive to it. You would just have to be a fly on the wall in some of these doctor's offices to believe what we have had to put up with all because we saw our children react to vaccines.

Kent is convinced, I don't know what to think. Sometimes I am so on board,and others I don't know, but most of the time I just dispare.

I have three family members that reacted to vaccines, all with acquired mitochondrial cytopathy.
Son reacted three different times to a DPT shot and the reactions were much worse than 105 temp, but a swollen left heart ventricle, a stroke, epilepsy, tourettes, and a high functioning autism. That was 25 years ago. He seems to just hate to move - and it is more than autism or brain injury because put him on adderol and he is as sharp and fast and helpful as they come.

My daughter also reacted to her vaccines. She had 105 temp, passed out gasped for air, and then came down with Kawasakis. It changed her personality, she had depression as a child, she had extreme depression as a teenager. She developed bipolar in her twenties with the help of a Hep b and flu shot (became a nurse and it is required) but it also helped push her into psychosis. This has been going on for 30 years now.

My poor husband, sick since a tetanus shot at age 34. I took him to Lowes today and he slept the whole way over there and the whole way back. He was a polmer research chemist with Dow chemical but he had to take disabilty have 29 years. 29 years in which he worked sick, taking lots of tramadol for muscle pain, xanax for panic attacks.

I think we are running out of time for him. I do so depend on him, and so do enjoy his company.

So if it is a virus, if it is a easy virus to knock out, I hope it comes very soon.

Dear Kent Heckenlively,

I appreciate your effort to understand the disease that affects your family, and to reach out to scientists who might have useful information. That said, I know very little about autism, including possible genetic or environmental components.

I should also say that I interpret the bulk of the scientific data to date to support the conclusion that XMRV is not present in humans. It's not that I want this to be the case. Indeed, I and many other retrovirologists were excited that we might use our knowledge of retroviruses to help understand and treat prostate cancer/CFS/autism.

So when you say your family members have tested positive for XMRV, I am prone not to believe the accuracy of the results, but would be glad to be convinced otherwise. Where were these assays done? If virus could be grown in culture from your wife and mother-in-law, what was the sequence of the virus(es)? These days, it is so easy to sequence viruses to establish their identity, that such sequencing would be required to document the nature of the virus detected. If the virus(es) very closely match known isolates of XMRV, such as VP62 or 22Rv1 XMRV, or known mouse retroviruses, then the finding may be the result of contamination. But if the viruses show some diversity, indicative of virus evolution in the new human hosts, then perhaps your family members are indeed infected. Have these assays been independently verified by a second lab?

Despite what I have said on this thread, I think your investigation of a potential link of autism to vaccination is a reasonable effort. I predict that XMRV will not be the culprit, but perhaps another virus is involved. As for me, while I am interested in the eventual resolution of the XMRV story, the small amount of funding that I have to study XMRV is about to disappear, and we are winding down our effort in this research area.

I wish you and your family the best, and hope you take no offense at anything that I have said.

Dusty Miller

Dr. Miller:
Thank you for coming here to debate. It has been very eye opening.
Your intial response was good, well thought out and then you decided to do mud slinging.

You said: "Vaccines bad, polio and smallpox good. Thanks for helping me understand."

Shame on you, you know that is not fair. It is like saying we traded one disease for another, so you should be thankful!

Is trading diseases what it is all about????

By the way Jack and Natasha - thank you, thank you, thank you for all the information you gave not just to Dr. Miller but to myself.

Dear Dr. Miller:

I appreciate your continuing engagement with me on the question of whether infection by the XMRV retrovirus may be related to autism and the question of whether vaccines may play any role in this disorder.

I'm not sure if you've followed my writings on the question of the possible relationship of XMRV to autism so let me briefly recap the information known by my more regular readers.

My daughter with autism/seizures tested positive for XMRV through antibodies. My wife and my mother-in-law tested positive through culture. I have tested negative for the virus as has my father. My mother passed away fifteen years ago, but given the results with my father and I the more reasonable conclusion is that she would also have been negative.

This evidence actually mitigates against a belief that current vaccines contain XMRV, although leaving out the question of how my mother-in-law came to acquire the virus.

To tell you about my wife, as a teenager she had psoriasis so severe it covered 90% of her body. Although she has been a speech therapist for more than 20 years in the rehabilitation unit of one of our best local hospitals, for the past few years she's noticed significant changes in her cognition. "I test people for brain injuries," she tells me, "and if you tested me you'd find significant deficits." She compensates for them, but strongly believes testing would reveal them. My mother-in-law was one of the first 1,000 people in the United States diagnosed with celiac sprue disease.

I must stand with my fellow autism parents, though, in my assertion that there was a change in my child after a vaccination. However, there were also many other complicating factors. My daughter had just gone off of breast milk and was on a cow's milk formula. She was only six months old so it was difficult to assess development as my wife and I were also late developers. At ten months of age she was diagnosed with a seizure disorder and at the age of three she was diagnosed with autism.

I believe the milk allergy plays into this disorder as for reasons unrelated to autism my son was kept on a hypo-allergenic milk formula when we switched him at fifteen months to save some money. At his eighteen month check-up the pediatrician ran him through a full developmental work-up, he passed with flying colors, and he recieved his shots without me giving it a second thought. A few days later my wife mentioned that she hadn't heard our son speak in a day or two. He'd had 15-20 words on the day of the visit to the pediatrician.

By chance I ran across the book, "Unraveling the Mystery of Autism" by Karyn Serrousi which thought gluten and casein were at the root of the problem. I put my son and daughter on the diet the next day. After 12 days my son said his first word again. it took him about a year to catch up in his language development, and about two years for his sensory problems to go away. The diet did nothing for my daughter and since that time I've always wondered what process we arrested in my son, but failed to do with my daughter.

After the diagnosis of my daughter with the XMRV retrovirus I contacted the UCSF pediatric AIDS unit to discuss the issue. I mentioned only that my daughter had been tested positive for this retrovirus, and that she had autism, and that there was a possibility that the two conditions could be linked. The representative with whom I spoke immediately said, "Well, that would explain why vaccines might trigger autism." I was a little stunned by this comment and asked him to explain further explain himself.

He said that all one needed to do was liken what is done with children who are born to HIV infected mothers with the situation I described. Babies born to HIV-infected mothers are given anti-retrovirals in order to lower the viral load prior to giving any vaccinations. As I'm sure you know, retroviruses like to hide out in the B and T cells of the immune system so that any stimulation of the immune system has the possibility of stimulating the virus. Although he was just the media representative, he felt fairly confident that this was a basic tenent of retrovirology. I must also note that an allergy, and my wife's family has been plagued with milk allergies, is also a condition which will produce an inflammatory response.

Considering that at a rate of 1 in 100 children with autism, this disease is more than 20 times more prevalent that polio was during any epidemic. A recent study suggests that autism is not genetic, as has long been the orthodoxy, but is more likely related to environmental factiors, of which prenatal infections may be a significant factor. http://archpsyc.ama-assn.org/cgi/content/full/archgenpsychiatry.2011.76

The question of whether vaccines at present contain XMRV is an open one, but nobody can doubt that vaccines are designed by their very nature to stimulate the immune system and produce a mild inflammatory response. Thus, regardless of the source of XMRV, a vaccination has the potential to stimulate XMRV, just as a vaccination will stimulate the HIV virus.

The finding of 10 times more viral particles of an unsuspected porcine circovirus than of the target virus in the rotarix vaccine raises the question of other unsuspected viruses circulating in biological materials given to humans. It is my hope that advances in technology, like deep sequencing, will enable us to answer these questions and make chronic diseases as rare as infectious diseases are today.

All the best,
Kent Heckenlively

Dusty Miller: “OK, so I stepped into the minefield of vaccination. Vaccines bad, polio and smallpox good. Thanks for helping me understand.”

So this is the level of reasoning of someone in charge of a well-funded, esteemed science institution http://labs.fhcrc.org/miller/, someone at the helm of humanity’s progress and future health?

God help us all!


Per...............

Is the asteroid bigger than a bread box?

Posted by: Media Scholar | July 17, 2011 at 02:30 AM

I hope the asteroid is as big as North and South American in mass/size and is headed directly for the Earth. A bread box would burn up in our atmosphere and do nothing. I want to see something that would put the earth out of commission. No more vaccines, no more nothin'

OK, so I stepped into the minefield of vaccination. Vaccines bad, polio and smallpox good. Thanks for helping me understand.

Can we get back to the subject of the article by Zhang et al. that so scared Heckenlively: xenografting and the spread of XMRV-like retroviruses? Infection of human tumor cells by xenotropic retroviruses following xenografting (xenotransplantation) of the cells in mice has been known at least since the 1970's. Production of xenotropic retrovirus by New Zealand Black (NZB) mice has been known for a similar length of time. Inadvertent spread of retroviruses in cell culture has been found in several cases, published and unpublished. One can improve cell culture techniques to prevent virus spread in the lab, or simply destroy cell cultures that become infected.

In their article, Zhang et al. provide an update on the situation using current techniques for virus detection. They confirm the extent of xenotropic virus contamination of human tumor cells that have been grown in mice, and spread of these viruses to other cell lines. The report provides a useful reminder of the possibility of retrovirus contamination and spread, but is not unexpected based on much older reports. I find it interesting that the authors did not detect XMRV in other xenotransplanted cells lines besides 22Rv1, which supports the Pathak/Coffin et al. conclusion that XMRV arose from a very rare recombination event. However, none of this justifies Heckenlively's description of this article as the "scariest reading of the year".

I concede to points raised in several posts that it can be difficult to entirely rule out the presence of known and unknown viruses in vaccines. Obviously, there are good examples of failures in this regard.

Oh, I feel so much better that the manufacturers and the FDA are going to the greatest lengths to making sure that the vaccines are not contaminated with other retroviruses. Thank you Dr. Miller. Oh, wait a minute. Did not the porcine retrovirus sneak in and in large quantities in the rotavirus vaccines? Yes, they did miss that one. It was only because some independent lab with "new" technology found it that we know now that the vaccine was contaminated. So they pulled one vaccine off the market and kept giving the other to small babies. Then, when they finally discovered that the other vaccine was contaminated with both versions of the porcine retrovirus, they decide to put both vaccines back on the market knowing full well that they contain the porcine retrovirus. Nice.

Perhaps Dr. Miller might want to comment on how the rotarix vaccine ended up with 10 times more porcine circovirus fragments than the target virus for the vaccine. As other commentators have suggested, if you don't know what you're looking for, it may be difficult to find.

This is from "This Week in Virology" by Dr. Vincent Racaniello, March 29, 2010.

"How did a porcine virus contaminate Rotarix, which is produced in Vero cells? The answer is not known, but the authors speculate that the culprit might be porcine trypsin, which is used during the propagation of Vero cells. Over 100,000 porcine circovirus 1 DNA molecules were detected in each vaccine dose, fully 10 times higher than the amount of rotavirus present. However, it’s not known if the porcine circovirus present is infectious."

P.S.

regarding your comment: "And certainly, any cell line infected with XMRV would not pass testing required for use of the cells in vaccine manufacture. Part of the reason is because XMRV replicates so well in many cell lines that it is easy to detect by standard reverse transcriptase assays"

Are you forgetting that a virus may well be latent and not actively replicating? Are you forgetting that xmrv was discovered decades after vaccines and other biologicals have been in use? Also worth remembering instances when RT activity was detected in the past in cell lines used for production of biologicals, but was deemed (by regulators and manufacturers) to "present no danger". Even though they would not have a clue as to its source...


---------

FDA Letter to Viral Vaccine IND Sponsors - Use of PCR-based Reverse Transcriptase Assay (re detection of retroviruses in their products), dated 1998

http://www.fda.gov/BiologicsBloodVac.../ucm105911.htm

… Whichever PBRT test you choose to employ, its performance should be validated, especially with regards to the lower limit of detection, the specificity (generality for retroviral RTs, negative for non-retroviral polymerases with RT activity), and the reproducibility of the assay. The lower limit of detection ("sensitivity") should be comparable with the published literature. ... it is possible that the results will be positive, which may require further characterization of the source of the positive activity. In the latter case, IND sponsors are recommended to contact CBER for further guidance in this regard, prior to undertaking any additional testing. …. If positive results are obtained, infectivity studies may be requested to demonstrate that the source of the positive activity is not an infectious retrovirus.

“are recommended”, but not legally required? What would “infectivity studies” be? Would those studies definitely rule out XMRV or any novel/still unknown exogenous infective retrovirus? Infectious ALV HAS BEEN detected in vaccines, and deemed to present no danger? Reassuring? What about such testing in 30's, 40's, 50's?

Dr Miller,

I would have bet my life that you would come up with the following line sooner or later (isn't it funny, you all do, always :)

"But what about vaccines? Yes, there is the possibility of undetected viruses in vaccines, but vaccine manufacturers and the FDA go to great lengths to avoid this possibility. And certainly, any cell line infected with XMRV would not pass testing required for use of the cells in vaccine manufacture. Part of the reason is because XMRV replicates so well in many cell lines that it is easy to detect by standard reverse transcriptase assays, which measure an enzyme that is present in all retroviruses."


That has become a sort of mantra, but may well be miles away from what really happens. These two documents (there are many others) illustrate the trap nicely:


NOTE FOR GUIDANCE ON QUALITY OF BIOTECHNOLOGICAL PRODUCTS: VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN (CPMP/ICH/295/95)

http://www.ema.europa.eu/pdfs/human/ich/029595en.pdf

3.2.1 Tests for Retroviruses
For the MCB and for cells cultured up to or beyond the limit of in vitro cell age used for production, tests for retroviruses, including infectivity assays in sensitive cell cultures and electron microscopy (EM) studies, should be carried out. If infectivity is not detected and no retrovirus or retrovirus-like particles have been observed by EM, reverse transcriptase (RT) or other appropriate assays should be performed to detect retroviruses which may be noninfectious. Induction studies have not been found to be useful.

… Cell lines derived from rodents usually contain endogenous retrovirus particles or retrovirus-like particles, which may be infectious (C-type particles) or non-infectious (cytoplasmic A- and R-type particles). The capacity of the manufacturing process to remove and/or inactivate rodent retroviruses from products obtained from such cells should be determined. ...


6.4 Limitations of Viral Clearance Studies
Viral clearance studies are useful for contributing to the assurance that an acceptable level of safety in the final product is achieved but do not by themselves establish safety. Number of factors in the design and execution of viral clearance studies MAY LEAD TO AN INCORRECT ESTIMATE OF THE PROCESS TO REMOVE VIRUS INFECTIVITY. These factors include the following:

1. Virus preparations used in clearance studies for a production process are likely to be
produced in tissue culture. The behaviour of a tissue culture virus in a production step
may be different from that of the native virus; for example, if native and cultured
viruses differ in purity or degree of aggregation.

2. Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid
initial phase is followed by a slower phase. It is possible that VIRUS ESCAPING A FIRST INACTIVATION STEP MAY BE MORE RESISTANT TO SUBSEQUENT STEPS. For example, if the
resistant fraction takes the form of virus aggregates, infectivity may be resistant to a
range of different chemical treatments and to heating.

3. The ability of the overall process to remove infectivity is expressed as the sum of the
logarithm of the reductions at each step. The summation of the reduction factors of
multiple steps, particularly of steps with little reduction (e.g., below 1 log10), MAY OVERESTIMATE THE TRUE POTENTIAL FOR VIRUS ELIMINATION. Furthermore, reduction values achieved by repetition of identical or near identical procedures should not be included
unless justified.

4. The expression of reduction factors as logarithmic reductions in titer implies that,
while residual virus infectivity may be greatly reduced, IT WILL BE NEVER REDUCED TO ZERO.
For example, a reduction in the infectivity of a preparation containing 8 log10 infectious units per ml by a factor of 8 log10 leaves zero log10 per ml or one infectious unit per ml, taking into consideration the limit of detection of the assay.

5. Pilot-plant scale processing may differ from commercial-scale processing despite care
taken to design the scaled-down process.

6. Addition of individual virus reduction factors resulting from similar inactivation
mechanisms along the manufacturing process may MAY OVERESTIMATE OVERALL VIRAL CLEARANCE.

and

Limitations of the process to remove infectious viruses from cell lines

www.emea.europa.eu/pdfs/human/bwp/026895en.pdf
Note points 1.6 and 1.7 on page 3 – I could not copy and paste…

On page 5 they say, under 3.2:
"Results have shown that EVEN SMALL MODIFICATIONS IN PROCEDURE OR THE PARTICULAR LABORATORY STRAIN OF VIRUS USED CAN HAVE A LARGE EFFECT ON VIRUS REMOVAL OR INACTIVATION"

also note 3.5 "occasional cases have been reported" (what about unreported ones?)

On page 6 they talk about rodent retroviruses. This guideline was written in 1994, what was the knowledge/awareness before then?

Interesting discussion on page 9 on "Limitations of Validation Studies", i.e. numerous factors that may lead to INCORRECT ESTIMATE OF THE ABILITY OF THE PROCESS TO REMOVE NATURALLY OCCURRING VIRUS INFECTIVITY


D Miller,

There isn't anything wrong in your reply on the surface, but the reality is while that yes vaccine makers and the FDA do go to great lenghts to detect viral contamination (I should know- I worked in vaccine development and manufacture for more than a decade and know this first hand) you can't test for what you don't know about. These tests are specific for certain known viral contaminants. So, no matter how hard you try there is no certainty that you have a culture free of viral contaminants.

And yes, with good laboratory practices you should easily control contamination. But all it takes is one time and your culture is screwed. Working all day long everyday I'm willing to bet some 24 year old lab tech in the middle of switching playlists on his ipod will inadvertantly mix up spent media with fresh media. It happens. I've seen it happen again and again. Humans are careless.

Oh how nice of Dr Miller to go out if his way to reassure the silly us. Perhaps he might also want to reflect on this paper:


Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals

J. Shepherd et al, Biologicals, Volume 31, Issue 4, December 2003, Pages 251-260

Rodent cells are used widely to manufacture recombinant proteins for pharmaceutical use in humans and animals. However, all rodent cell lines express endogenous retroviruses that require appropriate testing regimes for identification and characterisation. In this communication we report the results of transmission electron microscopy, reverse transcriptase assay and infectious virus assays for retrovirus in 185 manufacturer cell banks of mouse, rat or hamster origin. The results indicated considerable variability of retroviral expression levels by transmission electron microscopy and reverse transcriptase assay, but nevertheless characteristic features of each cell type were observed. INFECTIOUS RETROVIRUS WAS DETECTED IN MOUSE MYELOMA AND HYBRIDOMA CELL LINES, but not in cell lines of hamster or rat origin...

Rodent cell lines have for many years been used as substrates for production of biological therapeutics such as monoclonal antibodies, recombinant proteins, vaccines and gene therapy virus vectors. It has long been recognised that such cell lines contain retrovirus elements that may be expressed as particles detectable by electron microscopy. Such particles may be INFECTIOUS, AS IN THE CASE OF MURINE LEUKEAEMIA VIRUS (MLV), or defective and non-infectious, as in the case of the Chinese hamster ovary (CHO) cell retrovirus. DESPITE the lack of evidence for an association between murine retrovirus and disease in man, the POTENTIAL contamination of therapeutics with agents associated with oncogenicity and immunosuppression in therapeutic products is a cause of regulatory concern...

"regulatory concern" - sounds very reassuring, doesn't it?

This illustrates is how concerned our regulators really are:

document (2004) from European Medicines Agency’s CONCEPT PAPER ON THE DEVELOPMENT OF A GUIDELINE ON VIRAL SAFETY EVALUATION OF BIOTECHNOLOGICAL PRODUCTS TO BE USED IN CLINICAL TRIALS states the following:

… Validation studies are complex and expensive and a clear position on this is important. The extent to which manufacturers are able to refer to in-house experience concerning virus safety evaluation. For such cases, the virus safety of a given cell culture system and/or the capacity of established manufacturing process steps to inactivate/remove potential virus contaminants may have been demonstrated with several previously developed products. Such a database may serve as supportive data to JUSTIFY A REDUCED VIRUS SAFETY EVALUATION PROGRAM FOR NEW PRODUCTS THAT ENTER DEVELOPMENT.


let me repeat that last bit: "Such a database may serve as supportive data to JUSTIFY A REDUCED VIRUS SAFETY EVALUATION PROGRAM FOR NEW PRODUCTS THAT ENTER DEVELOPMENT."

Reassuring, hey!

Here are my comments about Heckenlively's post:

Re: XMRV -Science Journals
Scarier than Science Fiction

This piece by Kent Heckenlively confirms that a little
bit of knowledge can be dangerous!

The problem with the piece is that he got the wrong
definition of "xenotransplantation".

Xenotransplantation refers to transplantation of cells
between species, not just from mice to humans, as
Heckenlively quotes.

In fact, the report he discusses deals with
xenotransplantation of human cells into mice, not the
reverse.

This procedure is used to coax human cancer cells to
grow outside of humans for further study.

Surprisingly, human cancer cells are often difficult to
grow in cell culture, outside of a human, but are more
easily grown in nude mice.

While mouse cells have been transplanted into a few
humans, this procedure is very rare and could in no
way explain chronic fatigue syndrome or autism.

A. Dusty Miller, PhD


Heckenlively responds with the comment:

"I understand a Dr. Dusty Miller from the Fred Hutchison Cancer Center has raised some questions as to my suggestion that mouse tissue which harbors XMRV may possibly end up in a vaccine."


Nowhere did I mention vaccines. Neither did the paper Heckenlively finds so scary.

But what about vaccines? Yes, there is the possibility of undetected viruses in vaccines, but vaccine manufacturers and the FDA go to great lengths to avoid this possibility. And certainly, any cell line infected with XMRV would not pass testing required for use of the cells in vaccine manufacture. Part of the reason is because XMRV replicates so well in many cell lines that it is easy to detect by standard reverse transcriptase assays, which measure an enzyme that is present in all retroviruses.

What about retrovirus spread in the lab? XMRV is not like the ghouls from the Night of the Living Dead, able to be shot and still pop up to attack. XMRV and other retroviruses are fragile, such that treatment of these viruses for a few seconds with detergent or 70% ethanol, common laboratory disinfectants, kills them very effectively. However, culture medium from infected cells can contain over ten million infectious virus particles per milliliter, so one does need to be careful to prevent virus spread. For example, feeding infected and uninfected cultures with the same pipette can easily spread the virus. But with proper care, it is simple to keep XMRV and other retroviruses under control.

I agree totally. Especially the FedUp comment. We are living on borrowed time and one day soon it could very well happen with no warning at all. If the dinosaurs could be extinct with no warning, we shouldn't be so conceited it won't happen to the human race especially with what Kathy Blanco is saying. Since vaccine and drug manufacturers are doing an excellent job at laying waste to the human race; the final kicker with an asteroid hit wouldn't be so bad, come to think of it.
----------------------
Is the asteroid bigger than a bread box?

In the 1990's I use to show my students a video were a top scientist that worked for the CDC would fly all over the United States to look for the hantavirus. He would come in looking like a great hunter/warrior/ doctor wearing his protective gear hmmmmmmm made the girl's in my class hearts all go pitter patter, and the boys green with envy.

So, where might this great warrior/hunter/doctor of mouse viruses from the CDC at now????

This would be a great article for the Huffington Post. It's so revealing. I have read several parents' posts on the autism recovery forums, that their children tested positive for microbes found in dogs and cats and in the vaccines given to dogs. Do you think these viruses you speak of can also be transmitted from cats to humans via a bite. After all, cats eat mice.

the truth will never ever be told, it has been a complete cover-up since day one...anytime any researcher got close to any answers the carpet was pulled from under them...there is no doubt in my mind that 45% of the hiv virus envelope will be a major step to the truth...in 1969 25 million u.s. dollars changed hands between the dept of science and technology and the u.s. military and low and behold the hiv epidemic started years later and the exact words used in that meeting were immuno-deficient and immuno-compromised where one will kill the host and the second will incapacitate the individual...we are all led to believe that m.e. has been around for a century but that is an outright lie, cfs is a new entity created in a u.s. lab...hiv virus is not naturally occuring, it is a synthetic creation...xmrv is a total waste of time and is would only stand to cover-up the full truth from emerging...we know that u.s. troops were vaccinated before iraq and we also know the threat imposed by suddam if they were to invade iraq and the u.s government knows what suddam had as a weapon because it was the u.s who armed suddam with these weapons prior to iraq invasion...this is exactly why the cdc/nih have stalled for decades on cfs and including autism and they will continue to do so...they will alwys come out and say they care and in the end it is all part of the military bio-warfare that was created...i will never believe in all the hype that comes from especially cdc/nih...they are ruthless.....sincerely and always the truth aidan walsh southampton, u.k p.s. xmrv is probably a result of a piece of the aids virus.....

WOW! I knew that i was REALLY sick every day for the past 24 years, but this is SCARY!" xo TMH

The best-laid schemes o' mice an' men
Gang aft agley,
An' leave us nought but grief an' pain,
For promis'd joy!

Burns, R (1785) "To a Mouse, on Turning Her Up in Her Nest with the Plough" http://en.wikipedia.org/wiki/To_a_Mouse

Dr. Miller has placed those that should be inmates in charge of the asylum ..new meaning on three blind mice..cant see because of the $$$$ signs $$$$

Great article

Angus

Isn't it *odd* that Vincent Racaniello chose to kill off his mice after the xenograft study was released online?

"Transgenic mice susceptible to poliovirus
Posted: 12 Jul 2011 07:32 AM PDT
Yesterday I terminated the last remaining mice in my small colony, including the line of poliovirus receptor transgenic mice that we established here in 1990. Remarkably, I had never written about this animal model for poliomyelitis which has played an important role in the work done in my laboratory.

While I was still working on poliovirus as a postdoctoral fellow with David Baltimore, I became interested in how the virus causes disease. There were no convenient animal models to study poliovirus pathogenesis, so I began to think about the cellular receptor for the virus and how it could be used to make a mouse model for infection. When I moved to Columbia University Medical Center in 1982, I decided to identify the cellular gene for the poliovirus receptor. This work was carried out by the second graduate student in my lab, Cathy Mendelsohn. She identified a gene from human cells that encoded a protein which we believed to be the cellular receptor for poliovirus. When this human gene was expressed in mouse cells, it made them susceptible* to poliovirus infection (the mouse cells were already permissive for poliovirus replication). The gene encodes a transmembrane glycoprotein (illustrated) that we called the poliovirus receptor (PVR), later renamed CD155. Over the years we worked extensively on PVR, with the goals of understanding its interaction with poliovirus during entry into the cell. In one project we collaborated with Jim Hogle, David Belnap, and Alasdair Steven to solve the structure of poliovirus bound to a soluble form of PVR. The image of that complex decorates the banner at virology blog and twiv.tv.

Shortly after identifying PVR as the cellular receptor for poliovirus, a new student, Ruibao Ren, joined my lab. For his project I suggested he create transgenic mice with the human gene for PVR. We already knew that synthesis of PVR in mouse cells allowed the complete poliovirus replication cycle. Together with Frank Costantini and JJ Lee, Ruibao produced PVR transgenic mice and showed that they were susceptible to poliovirus infection. The illustration at top left shows a PVR transgenic mouse with a paralyzed left hind limb after poliovirus inoculation.

Poliovirus transgenic mice were used for many years in my laboratory to study how the virus causes disease, and to identify the mutations that attenuate the neurovirulence of the Sabin vaccine strains. A good summary of this work can be found in my review, ‘One hundred years of poliovirus pathogenesis‘. But there is a dark side of this story that I wish to briefly recount. When we first developed PVR transgenic mice, my employer decided to patent the animals. Until the patent issued, we could not share the transgenic mice with other researchers. As a consequence, others developed their own lines of PVR transgenic mice. One of these lines has been qualified by the World Health Organization to determine the neurovirulence of the Sabin vaccine strains. However, Columbia University realized little income from the PVR transgenic mice – such animals cannot be patented in Europe. By patenting the mice, we simply delayed research progress. Because of this experience I am personally very wary about patenting biological discoveries.

There are several reasons why I decided to stop doing research with mice. The cost of housing and breeding mice is very high, nearly $1.00 US per cage per day, and I simply don’t have the funds to support such work. More importantly, no one in my laboratory has any interest in working with mice: the last student to do mouse work left years ago. Although there are many interesting experiments to be done using viruses and mice, that line of work ended for the Racaniello lab on 11 July 2011.

*A susceptible cell bears the receptor for the virus; a permissive cell allows viral replication. A susceptible and permissive cell allows the complete viral replication cycle.

Ren, R., Costantini, F., Gorgacz, E., Lee, J., & Racaniello, V. (1990). Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis Cell, 63 (2), 353-362 DOI: 10.1016/0092-8674(90)90168-E"

Thank you Kent. Imagine if the same mobilization campaign happened for XMRV testing as is done for flu shots. I am chagrined that every effort seems to be made to avoid discovering the scope of the problem.

The International ME Association has also posted an answer to Dusty Miller's response at http://www.mecfsforums.com/index.php/topic,8102.msg98678.html#msg98678

"Dr Miller demonstrates the danger of an overreliance on metaphors in the guise of definitions. The study (Zhang, 2011) demonstrates how recombinant MLV viruses have spread from mice into cell lines and tissue cultures. The study further demonstrates that X-MLVs can enter other cell lines being held in the laboratory, at the same time. These recombinant X-MLVs are highly infectious and hence the process of xenografting can easily explain the entry of a number of related but different X-MLVs into the human population. The presence of these viruses could in every way explain the aetiology of ME/cfs and autism"



Per...............

Our scientific arrogance has now reached an all time high. A sure sign we are overdue for an asteroid impact.

Posted by: FedUp | July 16, 2011 at 06:56 AM

and..................

witches brew now takes on a new meaning. Green that vaccine? Not possible. One or two ingredients, is not greening a vaccine. Especially if they still use animal and human cell lines. I am now wondering if the simean CMV virus found in polio has helped the retrovirus cauldron even more? Since I am positive per WPI, and my kids with autism, the bigger question is, how do we get out of this fine mess? HIV children often have neurological problems very close to autism, hmmm?

Posted by: kathy blanco | July 16, 2011 at 11:54 AM

I agree totally. Especially the FedUp comment. We are living on borrowed time and one day soon it could very well happen with no warning at all. If the dinosaurs could be extinct with no warning, we shouldn't be so conceited it won't happen to the human race especially with what Kathy Blanco is saying. Since vaccine and drug manufacturers are doing an excellent job at laying waste to the human race; the final kicker with an asteroid hit wouldn't be so bad, come to think of it.

An excellent report, thank you.

Bear in mind that the US and UK government Health services have been actively blocking any research into ME/CFS since 1985. A retrovirus was found in us in the early 1990s and that work was suppressed.

Please help us by calling our illness ME/CFS. The other way around is what the psychs are using in an attempt to hang on to the derogatory name of CFS.

"With this recent finding regarding the dangers of XMLV and XMRV transmission in labs, the question becomes, 'Will these same medical authorities screw the very people who work for them?'"

Obviously, the answer is yes. If they don't mind making everyone else sick, why should they mind making their employees sick. But this may be what ends their reign of terror. If they make these lab workers sick, their "secrets" may be made public. I wonder if they've thought of that.

Thank you, Kent, for posting this.

Patricia Carter

witches brew now takes on a new meaning. Green that vaccine? Not possible. One or two ingredients, is not greening a vaccine. Especially if they still use animal and human cell lines. I am now wondering if the simean CMV virus found in polio has helped the retrovirus cauldron even more? Since I am positive per WPI, and my kids with autism, the bigger question is, how do we get out of this fine mess? HIV children often have neurological problems very close to autism, hmmm?

Wow, Kent, this is my worst nightmare scenario. Just what I was afraid of. Great article by way, and again, thank you so much for raising awareness on XMRV!

Since there is no possibility for those responsible for this horror to deny that these MLVs are indeed contaminating many cell lines and have done so for a few decades without anybody noticing, their only way to protect their reputation (and $income$) is to claim that these MLVs are not associated with disease, and that MLVs found in patients' samples are just the rsult of contamination.

This comes with the added benefit of destroying the reputation of the brave researchers who first discovered XMRV and associated it with diseases (prostate cancer, CFS, etc...), and scaring other researchers from trying to reproduce their results.

Deja vu?

And meanwhile, the horrifying truth is that these MLVs keep getting into the population through injection of biological products and through vertical transmission from infected mothers to their babies.

This could be all preventable. If these retroviruses let loose into the population are reponsible for the autism epidemic (and other modern epidemics), actions could be taken now to prevent new babies from being infected.

Yet nothing is done and this is beyond criminal.

Postscript:

Which means to them that the autism epidemic -- having humans with communication problems is perhaps not such a bad thing.

"Laboratories working with xenograft cultures should have full knowledge and understanding of the potential biological and biohazardous risks and should not distribute or publish their findings without full disclosure of the virus status of their xenograft-derived materials."

I am sorry Kent - can you translate what this means.

Thanks for translating the rest of it for me and us.

Just an after thought but,
I think the best bill we could ever pass since the orginally constitution is that all laws, lawyers, science and science papers must be written in simple language or not written at all.
But since lawyers and scientist are running this country and wants to keep being important, keep their power, and keep being in charge - they will keep the language as difficult as is nessacary.

Our scientific arrogance has now reached an all time high. A sure sign we are overdue for an asteroid impact.

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