Contamination is DOA

The recent paper by Christine Kozac traces the origins of the XMRV virus and she kills the contamination theory dead. She pretty much follows the MLV virus from around 8 to 12 million years ago or MYA to the present and shows how XMRV arrived in it's present form. The three things that this paper does and it's like the holy grail as far as I'm concerned. Christine Kozak goes on the refrigerator "ABOVE" Dr. Signh and Dr. Mikovits. Now why would I elevator her over Dr. Signh who is so much cuter? (grins)

1-She puts the "contamination question to bed". Period, end of story, any scientist who can read will know this is the end of the line for contamination silliness.

2-She shows that this virus originated around 100 to 130 years ago. Which I blogged about last year and she has confirmed (grins) and my ego is just big enough to really enjoy that. (HUGE GRINS)

3-She's produced a solid primer for researchers about what the potential problems are in regards to this virus. She shows what paths researchers need to follow up on. She shows that this virus has all the markers to produce pathogenesis in lymphocytic leukemia, erythroleukemia, immunodeficiencies, and neurological diseases.

I've laid it out with some quotes and page numbers for anybody that wants to slog through this instead of the 30 or so pages of the paper. Any misinterpretations are mine and mine alone and maybe the result of stupidly, brain fog, or lack of working brain cells. Any mistakes that you find please let me know and I'll correct them.

The paper really does start from the beginning, not as far back as the primordial ooze but close in human terms, (grins) She follows the rise of "XMRV" from 12 Million Years Ago (MYA) to present.

She starts out showing how the receptor (the old XPR1) arose within the species of mice. You have to get to that receptor in order to have XMRV. She knocks out an entire range of mice right out of the gate.

The host range of these 3 MLV subtypes maps to the receptor binding domains
(RBDs) of their envelope (Env) glycoproteins, and their RBDs govern the ability of these
viruses to interact with their cognate receptors (page 4)
Meaning there are three types of MLV's that have developed in mice. They can be grouped according to the receptors or their ability to bind to cells their RBD's. E-MLV's or ecotropic MLV'S just infect mice and sometimes only the species of mice that they come from. These have an mCAT-1 receptor and are not a problem for humans since we don't have cells that would allow this to bind.

Both PMLV's and XMLV's CAN have the XPR1 receptor that would allow it to bind to human cells and to a range of animal cells but it turns out it's not that easy. There are other factors. First of all many of the virus's are Endogenous so they are bound into the DNA of the mice they are in and don't produce any infectious virus.

Polytropic MLV ERVs (M/Pmvs) There are up to 40 copies of P-MLV ERVs in the laboratory mouse genome. The P-MLV ERVs have been divided into two closely related subgroups that differ most notably by the presence or absence of a 27-bp segment in the proline rich domain of env. These 2 P-MLV ERV groups are termed polytropic (Pmvs)

Although the coding regions of many M/Pmvs have open reading frames [25], none are
apparently capable of producing infectious virus; the reason for this is unknown, but
may be due to accumulated mutations

[However,] replicating E-MLVs can recombine with M/Pmv ERVs in mice to produce recombinant viruses with M/Pmv env sequences; these viruses generally have polytropic host range,(page 5 and 6)

In the apparent absence of recombination, the transcribed products of M/Pmvs can also be packaged as homodimers into virions of exogenous ecotropic virus, and these mobilized M/Pmvs can infect cells, replicate in those new cells, and spread to other cells as pseudotyped virus. Another transmission mechanism allows P-MLVs to completely bypass the need for their cognate receptor. These viruses are able to use alternative receptors in
the presence of the soluble RBD glycoprotein for that receptor. Thus, entry defective EMLVs
as well as P-MLVs, but not X-MLVs, can be transactivated in this way by E-MLV

However it turns that the nice quite little viruses can, when they come into contacts with other Ecotropic or mouse only virus's, wake up and get busy making a working virus that has the ability to by pass certain restrictive mechanisms and produce a variant [B ]infectious virus
. This possibility was discussed in the science community early on regarding XMRV however, it looks like "recombination" is not an issue for the XMRV virus. It looks like it evolved over time to be a straight up novel Exogenous virus.

The XMLV's break out further into groups and are a bit newer on the phylogenetic tree. These virus's break out into three "clades" or subgroupings. Most do not produce infectious virus however within the clades or subgroups there are two that do produce infectious virus the NZB and the F/ST. It is interesting to note however that if certain chemical or bacterial events occur that there are among the non infectious clades or subgroups the ability to produce infectious virus. It doesn't say particularly what these event's might be but the paper treats these as of no importance to the development of XMRV itself.

two strains, NZB and F/St, have a high virus phenotype, producing high titers of X-MLV throughout most of their lives. Other strains rarely produce infectious virus, but cells from many common strains can produce virus following chemical induction or stimulation of spleen cells by bacterial lipopolysaccharide (LPS) or in a graft versus host reaction
I'm not sure if it is only in the laboratory or if events of this nature can occur in the wild. (oh, goody more bed time reading) So far all of the virus types that have been discussed can be found in both wild and laboratory mice and have been studied in the laboratory since the 1950's.

I think that's why she sort of stops on page 9 and discusses the problem of contamination. She sites several cell lines that are used to produce a variety of products that have mouse DNA in them and so have virus wrapped up in the DNA. As discussed above some of the quite XMLV can become active if you introduce certain chemicals or bacteria into them. (Note: This is speculation on my part but it appears that many scientist who were not necessarily working with XMRV experiments themselves remembered this bit of information from school days and so jumped on the "could it be contamination" bandwagon, possibly because the ideal of a mouse based endogenous virus being able to infect humans seemed a little 'world is round' to them, grins)

However the above being said she goes on to complete the tracing of the phylogenetic tree of the very unique XMRV and puts the potential of contamination completely to rest. Read on. (if you're not bored, grins)

At this point she looks at the wild mouse and it's origin in the Indian subcontinent 8 to 12 MYA (million years ago) and that the MUS split off from other murinea animals like the hampster and the mink shortly after, around 7 MYA. MUS Then split into 40 subspecies of mice and 3 of those became house mice around 1MYA to 1/2 MYA (I'm gettin' there Mark I swear, grins) from there "fancy mice" or pet mice strains were breed by humans (Note: this goes back to around 1500 C.E. in China and spread to Victorian England where pet mice were very popular, that's just my reading and is not included in the paper) and from "fancy mice" laboratory mice were breed starting in 1905. It turns out that wild mice don't carry the particular type of MLV's that are found in house mice so the phylogenetic tree splits at 1 to 1/2 million years ago and begins to develop in the house mouse strain. Turns out that's why the wild type mouse mus pharia which is being brought into the laboratory in order to study lab virus's is susceptible to the XMRV virus. Note: mus pharia like to play dead and bite. I like these guys already!)

So the MLV's that gave birth to XMRV turn out to be a from house mice rather than wild mice. (no mouse hate for the local field mice please.)

Although inbred strains of laboratory mice tend to carry multiple
copies of both Xmvs and M/Pmvs, these virus subtypes are largely segregated into
different species in the house mouse complex (page 10). Sequences
related to the env RBD of M/Pmvs are found in M. domesticus of Western Europe, while
Xmvs predominate in M. castaneus, M. musculus and M. molossinus in eastern Europe
and Asia
(Figure 3). Use of probes from the LTR and from env segments that are
outside the RBD largely confirmed this pattern of ERV segregation in Mus species, and
found two polytropic subtypes, Mpmvs and Pmvs, in M. domesticus as well as evidence
of atypical, recombinant types in the various house mouse species
Mus is not native to the Americas, but was introduced with human travelers.
American house mice most closely resemble the western European M. domesticus in
that they lack Emvs and carry multiple M/Pmv ERVs and few or no Xmvs
She next notes that Asian mice tend to carry many of the XMLV's while the western European Mus domesticus carries the multiple PMLV's and at the time when M. domesticus was brought to America there does not seem to be an indication of the X/P hybrid that would give rise to XMRV yet. This would indicate that XMRV's developed well after the settling of America or after 1500 to 1600 C.E.

Then it get's interesting. . .

One exception to this is found in Lake Casitas, California, where mice carry multiple copies
of Xmvs and M/Pmvs [71]. These mice also carry an Emv subtype common to Asian
mice [71, 74]. LC mice may thus represent a natural hybrid of European M. domesticus
with M. castaneus mice that may have arrived in America with Chinese laborers and
Cargo (page 11)
Turns out there is one wild type mouse that produces both Pmv's as well as Xmv's and this is a cross breed found in California (hmmmmm, where was that first out break? Oh yeah in California, grins) This mouse is a cross between M. domesticus with it's multiple PMLV's and Japanese fancy mice which produce XMLV's. These mice where most likely brought over by Chineese and Japanese laborers to the West coast where they hooked up and produced the Lake Casitas, California mouse So we finally have a mouse that has the ability to make an XMRV type virus in the Lake Casitas or LC mouse. However these mice don't produce virus with the correct receptor but with a variation of the receptor called BXV1. Rather than the XPR1 which can get into a lot more cells in a lot more animals.

So far we've gotten our mouse to the west coast of America sometime between 1800 and 1875.

The paper then narrows the field to look at 4 types of things that must be in play for a virus to infect humans and are unique to each MLV.

The various X/P-MLVs isolated from laboratory and wild mouse
species differ phenotypically on the basis of host range, variable reactivity with anti-MLV antibodies, cross-interference, cytopathicity, and pathogenicity in mice.
So you not only have to have and XPR1 host range but a specific type of host range like XPR1n or XPR1(sxv) that seperates where a virus comes from and what it can do. (page 15) Also what "restriction factors" come into play like does your virus get smacked down by APOBEC3G but slide by Trim5 (alpha) then it could only arise from a cross of these viruses. Also a what type of proteins that your virus codes for since they are all different and are there deletions that can pick up junk on the envelope of the virus allowing it to hide. So starting at page 12 Ms. Kozak breaks down how the various mice in the laboratory stack up to produce the XMRV virus.

One such isolate, CasE#1 (or Cas E No. 1), was isolated from a wild-trapped California mouse [77]. It resembles P-MLVs in its ability to produce MCF-type foci and in its interference properties, but, like X-MLVs, it fails to infect laboratory mouse cells and has novel receptor requirements. Cz524 MLV was isolated from the wild derived M. musculus strain CZECHII/EiJ, and differs from both P-MLVs and X-MLVs in host range. The env genes of these two wild mouse isolates are not identical to laboratory mouse P-MLVs or X-MLVs, but are related to both.
Two known virus strains have been isolated that produce virus that have shared characteristics of both P and X MLV's however looking at the isolates neither produced XMRV because neither produced the correct receptor to bind with human cells.

The importance of the fact that Christine runs through just about every lab mouse strain over the next few pages and very carefully shows that while a variety of laboratory mice have the ability to produce XMRV which is a cross between XMLV and PMLV none have produced the strain of XMRV that we are seeing in humans. This puts the question of contamination to rest. You could say that this paper is what we have been waiting for. This is the paper we could/can toss into Dr. McClure's face and say "HA". Indeed anybody who want's to shout "contamination" could be effectively extinguished with this paper.

The paper goes on to the next level of XPR1 receptor alleles that are defined by the ability of a particular portion of the receptor ELC's 1 to 4. Turns out you have to have a very specific one in order to infect a range of animals.

Infectious virus related to X/P-MLVs has been isolated from human patients with
prostate cancer and chronic fatigue syndrome [115, 117, 118]. This virus, termed
XMRV (xenotropic murine leukemia virus-related virus), shows close sequence
homology with X/P-MLVs [114], uses the XPR1 receptor [115], and has xenotropic host
range [79]. The VP62 isolate of XMRV and the sequenced DG75 X-MLV genome [120]
show overall 94% sequence identity [114]. A more complicated picture emerges from
sequence comparisons of the XMRV coding and non-coding domains with
corresponding regions of X-, P-, and E-MLVs, as well as the active Bxv1 Xmv and a full
length Mpmv. While XMRV most closely resembles the X-MLVs in SUenv and LTR, it
shows greater identity to M/Pmvs in gag and pol (Table 4). This, coupled with the
recent finding of M/Pmv related env and glycogag sequences in human blood donors
and chronic fatigue patients [119] points out the need for further work to clarify the
evolutionary path linking the human and mouse viruses . . .
(page 19/20)
In seperating out XMRV from the herd Ms. Kozak shows that while XMRV shares specifics with both known X/P MLV's the overall sequence identity to known viruses that have been studies for years does not reach beyond 94%. When comparing the current copy of XMRV VP62 it has similarities to some X type, some P type and some E types virus's. This shows that it absolutely is not one of the laboratory derived virus's but may have passed through another/other animal(s) before making it's way back to humans. However she qualifies this as saying it's a possibility not a certainty and this is where the rubber pretty much leaves the road.

The XMRV virus and X/P-MLV sequences found in humans may have been
acquired directly from mice, or after transmission from mice to another species in
contact with humans. If there is direct transmission from infected mice, this could be
reflected in the geographic distribution of virus and/or receptor type in mice and the
worldwide incidence of prostate cancer. Studies have reported very different rates of
XMRV detection in prostate cancer patients (reviewed in [122]), and while these
differences may have technical explanations, it is also possible that some of these
differences are due to geographic differences in exposure to XMRV. The highest rates
of prostate cancer are found in the U.S. and lowest rates are found in Asian countries
like Japan, India and China [123]. Rates in Europe are lowest in Eastern European
countries. This distribution generally corresponds to the distribution of Xpr1 receptor
variants in mouse populations; the most permissive allele, Xpr1sxv, is found in high
tumor incidence areas, and the most restrictive allele, Xpr1m, is found in low tumor incidence areas.
What makes her think that it is possible that the virus has more than one host is that the receptors XPR1 (sxv), shows up in areas where there are a lot of cases of prostate cancer like the US and Western areas of Europe while the XPR1 (m) group is found in the areas where there is lower incidents of Prostate cancer like Asia, Japan, India and China. She makes a good point but this could be as much about the genetics of the people living in those regions as the receptors on the virus.

She could have a good point in that ME/CFS also shows up more prevalent in Western Europe, America and Australia so there may be some genetic typing much like HTLV which tends to only be pathogenic in the Pacific Rim and Africa.

So that's pretty much it. Nothing that we didn't really know but some really solid science. There is a bit more on potential pathogenic problems on pages 20 to 24 and then some about host restriction factors and recombination possibilities that round it out.


I knew this paper was significant and I am glad you took the time to explain it to us :D
What would we do without you, George? Thanks so much for distilling Kozac's paper. There's no way I would have understood it otherwise.
I want to add my thanks to George for the reading and the explanation. It sounds like this particular paper is of much greater importance than it appears to the lay reader.
Dang, I didn't think anybody would read it! (grins) Well, heck I'm glad other folks got something out of it too, cause I mostly did it to make it solid in my brain for when later stuff comes out.
Myra McClure is a fool. I hope she reads your excellent analysis George. Reads it and weeps.
George, What a wonderful analysis

I don't know if I am reading too much into the paper or grasping at straws here. One of the mouse types mentioned is the NZB. Further reading on that type returns the information that it was sent to the Otago Medical School in 1930. That's the same medical school that investigated the NZ Tapanui flu outbreak of ME in the 80's.

This page talks about a possible virus in these mice
and oddly enough there are papers on the NZB mouse by Dr L. O Simpson. I think that this may be Dr Les O Simpson who has been working on blood cell shapes in ME. He's described as a retired pathologist on one ME group and I actually met with him when he came to London once.
I read a little factoid in a book a few days ago that 8% of our DNA is from a variety of retroviruses that we have absorbed/survived. My jaw dropped open, and in the middle of the book shop shrieked to my partner 'If this is the case, why is CFS/ME and XMRV so inexplicable to scientists????' I fumed! Now this wonderful paper has arrived. Thanks for breaking this down George, much appreciated.
Well, I'll be darn ukxmrv, that's really interesting! Thanks for posting that tidbit. Thanks Snow leopard for posting the link to Christine Kozac profile. I sure hope we hear more from her in the future. (big grins for Nermalina) pretty name, yeah it's a bit of shock once you get into all the science. It seems like everybody must have known all along that this illness was retroviral but nobody had the ????ability, funding, something???? to find out which virus was responsible. Joe DeRisi is my Hero. (slobbery dog licks!)
Quote from 1970 paper explaining the origins of the NZB mouse and relatives

The University of Otago Medical School mouse colony
was formed with randomly bred mice brought to New
Zealand by Mr. W. H. Hall in 1930 from the Imperial
Cancer Research Fund Laboratories at Mill Hill, Lon
Looking back at the outbreak at the Los Angeles Hospital in 1934 leads me to one interesting experiment being carried out under the auspices of the University of Southern California around the same time.

Some of the staff of the Uni were involved in a cancer experiment from June until late August (ish) 1934 involving the Rife method. I don't want to start a discussion on the pros/cons as it is controversial and will take us away from the bits we are interested in.

From what I can glean from the internet a group of cancer patients (not from LA as far as I can tell, his contacts seemed to be in San Diego and Pasadena) was taken to a ranch in La Jolla to see if the Rife methods would cure them. This is of interest as some of the doctors reported to be involved in a sort of steering committee for this worked at the Uni of Southern California including the President who worked in LA.

Rife claimed that he had discovered a cancer organism or virus from human BC specimens and injected this into mice and rats to test his theory that it was communicable. He was working on this in California in the early 30's. He obtained breast cancer material from humans and was experimenting on rats and mice. At the time it was fashionable to look for a virus in humans that would cause breast cancer. When I looked at Imperial Cancer Research in the UK I can see the same ideas and claims.

I've not been able to find out exactly what happened in the Rife experiments except that he incubated his human breast cancer specimens in a pork solution and the used rats and mice to see if they developed tumours. No exact details (i.e. exact type of mouse) seem to be on the internet. Most of the web stuff is just a repeat of the same quotes.

What it shows is just how uncontrolled experimentation was in 1934 and how close to an original M.E. outbreak was an experiment that featured LA doctors, a "cancer virus" and patients /mice to experiment on.

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