1. Patients launch $1.27 million crowdfunding campaign for ME/CFS gut microbiome study.
    Check out the website, Facebook and Twitter. Join in donate and spread the word!
The ePatient Revolution
Ryan Prior shares his experience and his thoughts from attending the Stanford Medicine X Conference as he contemplates the rising of the ePatient Revolution ...
Discuss the article on the Forums.

Would the mystery VP62 XMRV virus please step forward?

Discussion in 'XMRV Research and Replication Studies' started by jace, Apr 20, 2012.

  1. jace

    jace Off the fence

    Messages:
    855
    Likes:
    170
    England
    http://www.me-advocacy.com/VP62_please_step_forward.html

    Genbank holds two VP62-XMRV full length sequences. One (DQ399707.1) is from the first XMRV paper, Urisman et al. (2006), the second (EF185282.1) is from Dong et al. (2007). The second is two partially overlapping sections of XMRV from VP62 that have been fused together.

    There is also however a mysterious third sequence.

    What makes this third VP62-XMRV (NC_007815.1) peculiar is why, on the 3rd July 2011, this sequence was update/replaced by a PreXMRV-1 sequence with the accession number NC_007815.2.


    The mysterious third VP62-XMRV sequence

    PreXMRV-1 was first mentioned in Paprotka (2011), and was speculated by those authors to be a parental virus to a hypothetical virus that would be several switching events away from being the consensus 22Rv1-XMRV virus - more on the issues around the missing env gene for the consensus 22Rv1-XMRV GenBank sequence can be found here.

    Interestingly this PreXMRV-1 sequence that replaced a VP62 sequence is identical to another PreMXRV-1 sequence (FR871849.1). Both are said to relate to Paprotka et al. (2011). They are both said to be from the same mouse hosts, NU/NU and HSD. And they both have the same identical date, 3rd June 2011.


    NC_007815.1
    http://www.ncbi.nlm.nih.gov/nuccore/NC_007815.2

    FR871849.1
    http://www.ncbi.nlm.nih.gov/nuccore/FR871849.1


    Two particular details on the entry for the PreXMRV-1 virus, which replaced the third VP62-XMRV virus, do stand out above many others.

    Firstly, the locus tag for the gag gene is called "pXMRV-1_gp1". pXMRV1 happens to be the name used for an XMRV plasmid. Is there any relationship?

    The second and most significant is for the locus tag on the env gene, which is called "XMVV62_gp4". This is of interest because it is the same name given to the locus tag on the env gene for the VP62-XMRV virus that this PreXMRV-1 virus has replaced.



    PreXMRV-1

    There have always been questions raised about the PreXMRV-1 virus due to the statement in Paprotka that indicated the use of more than one source for complete sequencing of this putative virus.

    The complete sequence of PreXMRV-1 was determined from the early passage xenografts, the NU/NU and Hsd strains, and the CWR-R1 cell line. (Paprotka, 2011)

    Questions have also been raised about the failure to provide any details on cloning and identification of PreXMRV-1, despite the inclusion of details for the virus named PreXMRV-2.

    "Cloning and identification of Prexmrv2. A fragment of mouse DNA containing the gag leader with the characteristic 24?base deletion, the 5 LTR, and flanking mouse DNA was amplified and cloned with the aid of the Genome Walker kit (Clontech). From the flanking sequence, primers C12_1f and C12_4r were developed (Fig. S2) and used in combination with the internal primers shown in Fig. S2 to amplify Prexmrv2 sequences for detection and sequencing." (Paprotka, 2011)

    Furthermore, there is the issue of genome integration of a particular PreXMRV-1 sequence, which appears to be different to these two identical PreXMRV-1 sequences, into a strain of mice not used to create the 22Rv1 cell line. Further details on this can be found here and here.


    Where did the 3rd VP62-XMRV come from?

    The date on this third VP62-XMRV sequence is the 8th December 2008. The study title is Urisman et al. (2006). It is said to have been a direct submission to the Genbank and part of the CONSRTM NCBI Genome Project.

    We have been unable to find if this sequence was ever claimed by a study, but believe that the first reference to it was in Smith et al. 2010. http://www.retrovirology.com/content/7/1/70

    Although on the VP62-XMRV entry it states that, This sequence has been updated, and despite on the PreXMRV-1 update it says that, this sequence version replaced gi:89889045, when attempting to access the gene or protein information for this third VP62-XMRV sequence, there is line that says, This record was discontinued.

    Considering that the record has the title of the Urisman (2006) paper entered into its details, and the update is for Paprotka et al. (2011) perhaps Dr Silverman, Dr Pathak and Dr Coffin should now explain why a PreXMRV-1 sequence has replaced a VP62-XMRV sequence and what the reasoning is for this replacement?

    Studies that we have found to have mentioned or used this third VP62-XMRV sequence are listed below. Use of this now obsolete sequence raises questions about the validity of data derived from it.



    STUDIES

    Desarrollo de un nuevo sistema de transferencia gnica basado en un gammaretrovirus humano.
    Daniel Cervantes Garcia
    http://eprints.uanl.mx/2687/1/Tesis_Daniel_Cervantes_García.pdf

    Susceptibility of the Human Retrovirus XMRV to Antiretroviral Inhibitors: Discussion Robert A Smith, Geoffrey S Gottlieb and A Dusty Miller http://www.medscape.com/viewarticle/729233_4

    PCR and serology find no association between xenotropic murine leukemia virus-related virus (XMRV) and autism
    Brent C Satterfield, Rebecca A Garcia, Fiorella Gurrieri, Charles E Schwartz. http://www.molecularautism.com/content/pdf/2040-2392-1-14.pdf

    Detection of XMRV in normal and tumor tissue of prostate cancer patients from the Southern United States is dependent on specific PCR conditions
    Bryan P. Danielson, Gustavo E. Ayala, and Jason T. Kimata
    http://ukpmc.ac.uk/articles/PMC3058280

    An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV
    Eiji Sato, Rika A Furuta and Takayuki Miyazawa
    http://www.retrovirology.com/content/7/1/110

    No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected Konstance Knox, Donald Carrigan, Graham Simmons, Fernando Teque, Yanchen Zhou, John Hackett Jr., Xiaoxing Qiu, Ka-Cheung Luk, Gerald Schochetman, Allyn Knox, Andreas M. Kogelnik, Jay A. Levy
    http://www.sciencemag.org/content/suppl/2011/05/31/science.1204963.DC1/1204963s.pdf

    Phylogeny-Directed Search for Murine Leukemia Virus-Like Retroviruses in Vertebrate Genomes and in Patients Suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Prostate Cancer
    Jonas Blomberg, Ali Sheikholvaezin, Amal Elfaitouri, Fredrik Blomberg, Anna Sjo ?sten, Johan Mattson Ulfstedt, Ru ?diger Pipkorn, Clas Ka ?llander, ChristinaO ?hrmalm, and Go ?ran Sperber
    http://www.hindawi.com/journals/av/2011/341294/

    Disease-associated XMRV sequences are consistent with laboratory contamination Stphane Hu, Eleanor R Gray, Astrid Gall, Aris Katzourakis, Choon P Tan, Charlotte J Houldcroft, Stuart McLaren, Deenan Pillay, Andrew Futreal, Jeremy A Garson, Oliver G Pybus, Paul Kellam and Greg J Towers
    http://www.retrovirology.com/content/7/1/111

    xenotropic gammaretroviruses and their XPR1 receptor
    Christine A Kozak
    http://www.biomedcentral.com/content/pdf/1742-4690-7-101.pdf The mouse

    Early Events in Retrovirus XMRV Infection of the Wild-Derived Mouse Mus pahari
    Toshie Sakuma, Jason M. Tonne, Karen A. Squillace, Seiga Ohmine, Tayaramma Thatava, Kah-Whye Peng, Michael A. Barry and Yasuhiro Ikeda http://jvi.asm.org/content/85/3/1205.full.pdf

    Reposted with permission from http://www.me-advocacy.com/VP62_please_step_forward.html Comments are enabled on the link.
     
    currer, Desdinova, Lou and 1 other person like this.
  2. Ecoclimber

    Ecoclimber Senior Member

    Messages:
    651
    Likes:
    1,137
    Mercer Island Wa
    jace, I'll get back to you with all answers to your questions
     
  3. free at last

    free at last Senior Member

    Messages:
    617
    Likes:
    124
    Not that i understood most of this, but the idea of a sequence being replaced by a prexmrv - 1 sequence, is enough to make me parenoid
    Hope the guy whos freinds with so many of these scientists has a reasonable explanation for this change. No doubt he will ?
     
  4. Ecoclimber

    Ecoclimber Senior Member

    Messages:
    651
    Likes:
    1,137
    Mercer Island Wa
    Updates to GenBank sequences are routine based on new data regarding protein annotation, etc. These updates are reflected in the suffixes .1, .2, .3 etc. Details of the generation of sequences from integrated viruses, such as PreXMRV-1 and -2 are often complicated, and may involve PCR amplification of several fragments of the virus, sequencing of the individual fragments, and stitching of the sequences together. These techniques can introduce error, but in general, are highly reliable for determining the sequence present in mice or other organisms.

    Some of the scientists have moved on to other research projects and are no longer investigating XMRV.

    Eco
     
    barbc56 likes this.
  5. jace

    jace Off the fence

    Messages:
    855
    Likes:
    170
    England
    Your post keeps changing, Eco.

    You still don't update one virus for a different virus, unless they are the same virus.


    So, are they the same virus?


    PreXMRV-1 and 2 are separate viruses, and PreXMRV-2 isn't included in this discussion so far.


    What was stitched together then? Is PreXMRV-1 and VP62 the same virus?


    So have they reliably determined that VP62 is actually PreXMRV-1? which has a truncated gag and therefore cannot be the Mikovits and Ruscetti sequences?
     
    asleep and garcia like this.
  6. Ecoclimber

    Ecoclimber Senior Member

    Messages:
    651
    Likes:
    1,137
    Mercer Island Wa
    In answer to these additional set of questions Jace, I believe it would be best to wait until Lipkin reports in at the completion of the XMRV/MLV study.

    Eco
     
    barbc56 and Kati like this.
  7. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

    Messages:
    970
    Likes:
    458
    Noosaville, Aust
    Why? Are saying that if the Lipkin study finds something, then the responses you provided are wrong, or, perhaps more likely, you would rather wait for the Lipkin study which will also not address these questions, but perhaps provide another vague "nothing to see here" conclusion? Science by consensus again? How does the Lipkin study close things in such a way that questions should not be asked, or for that matter answered?

    Seems that you either know the outcome of the Lipkin study, or know what it is going to be. Omniscience or contrivance? Odd how many of the negative studies were telegraphed as being negative by those in the know way before completion or publication.
     
    asleep and garcia like this.
  8. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

    Messages:
    970
    Likes:
    458
    Noosaville, Aust
    Not sure of context of this statement. They don't have to answer to questions about their previous studies? Viruses are jumping from animal to human all the time, yet a virus which is shown to be infecting human tissue is not a valid research avenue? Or there is no funding for XMRV? Valid reasons closing out research, but not for failing to correct unfinished or incomplete research which could answer vital questions about a potentially infective agent.

    Jace's questions strike at the heart of the argument that XMRV is a non-dangerous lab artifact in studies that were pivotal in the anti-XMRV consensus. If there is a flaw in that argument, Lipkin's study will not uncover it.
     
    currer, asleep and garcia like this.
  9. Ecoclimber

    Ecoclimber Senior Member

    Messages:
    651
    Likes:
    1,137
    Mercer Island Wa
    There is no point in this discussion until Lipkin publishes the research on the XMRV/MLV project. To do so would be a bit premature and a disservice to Lipkin's research. That is all I can say at this point.

    There are several researchers not involved in the project that have moved on to other research projects outside of ME/CFS. After Lipkin's releases the NIH research study, I am sure there will be amble opportunity to address any issues you or your colleagues may have.

    The NIH funding has move to Lipkin's research project. I do not know about the status of WPI or what they will be doing with their NIH grant funds which will most likely be hammered out in a court of law and which may take some time. I have no knowledge of what is taking place at WPI in regards to research and wish to remain as far away from that situation as possible.

    There are other research project out there. Montoya hasn't reported in. Kogelnick on his pilot program. The Light research project. I think Cort wrote an article on the front page at the beginning of the year on the various projects. I have no idea of any of the CAA projects as I don't follow them. I am focusing on a project that might shed some light on the subject.


    Eco
     
    barbc56 likes this.
  10. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

    Messages:
    970
    Likes:
    458
    Noosaville, Aust
    Apart from repeating your comments of earlier posts, you appear not to have met any of my points, other than to say you would not comment further, which is your perogative, of course. However I would be interested in hearing from what perspective you consider Lipkin's study would/might settle the issues highlighted in Jace's questions, as I am unaware of how, even remotely, he would be looking at the origins of XMRV, or of its so-called two-part precursors.

    I repeat, how does Lipkin's study, whether he finds a virus or not, meant to settle the Pre-XMRVs questions?

    None of those researchers you mentioned here were involved with the anti-negative push, but are involved in non XMRV research, and in well-travelled directions already ignored by the CDC; some would argue funds have been made available for these areas as a sop, or to obstruct any constructive XMRV research - certainly anything with the CAA's fingerprints near it would fall into this category. Concerning the so-called anti-XMRV researchers, it does appear that they only entered the area to put a halt to further research and once that objective appears to have been met, are quite happy to move on. It will be interesting if in the future some other cracks appear in the wall of denial whether the same anti-XMRV researcher brigade will be trotted out.

    I note there was no lack of funding for studies which set out to disprove XMRV, only for studies genuinely trying to further research in that area. Most, if not all of the negative studies were directed towards proving contamination.
     
    currer, jace, asleep and 2 others like this.
  11. garcia

    garcia Aristocrat Extraordinaire

    Messages:
    934
    Likes:
    103
    London, UK
    I think you mean ample.
     
    froufox likes this.
  12. August59

    August59 Daughters High School Graduation

    Messages:
    1,480
    Likes:
    405
    Upstate SC, USA
    I think Lipkin's research will be full of twist and turns, especially using the MASS TAG PCR in conjuncction with other test. I believe Montoya's pathogen test may pick up on something new to at least give us a new direction to to be looking a any way. To be truthful I hope any study would miraculiously give us what information we need, but I'm afreaid there is still a lot more information to be found.

    I think it would be great if Montoya finds something interesting to keep researchers interest up and I actually believe a lot of people are watching this study, but damn it is taking forever. I think Lipkin is going to change the way we have looked gamma retroviruses and the way we we look from now on. I just beleieve there is something missing from our research that will reveal gamma retroviruses to manipulate something that allows a different pathogen, virus or genetic pre-disposition to take advantage of one or more triggering mechanisms to start some type of chain reaction that will the same in some, but different in others leading to a good standard group of subsets that I don't believe can be denied.

    Is there anyone or even any researhers that believe there are not subsets among what is described as CFS and even ME today?

    I also think research is poised to make great strides in its capabilities to analyse things plus the lucky time when a product proves to to have very positive unintended results.

    My worst thought of anything right now id it could not be at a worst time when funding is being cut and it not affecting us. Our biggest advantage is having such a large population in the wotld involved being patients, care givers or relatives that our private funding can step up if economies can begin to turn upward, but that is a another story. I certainly got off subject, but I am at a point where I see my end. I hope the new discoveries can avoid anyone from spending a life of turmoil and agony. Suffering humiliation at the hands of their own doctors, families and friends. I hope the day comes when it treated no differently than a minor cold. I HOPE!!!
     
    currer, jace, ukxmrv and 2 others like this.
  13. Ecoclimber

    Ecoclimber Senior Member

    Messages:
    651
    Likes:
    1,137
    Mercer Island Wa
    Tying up up some lose ends, although probably not.

    There has been some discussion on the missing Env. In the sequence file for FN692043.2, the annotation for the envelope protein is missing. The DNA sequence is correct, and the envelope coding region is present, but there is no description of the Env coding region.

    To make sure the Env protein coding region is present, if you translate the open reading frame, you will find that runs from DNA base 5754 to 7688 and you will get the following protein (single letter amino acid code):

    FN692043.2, translated 5754-7688[/B]
    MESPAFSKPLKDKINPWGPLIIMGILVRAGASVQRDSPHQVFNVTWKITNLMTGQTANAT
    SLLGTMTDTFPKLYFDLCDLVGDNWDDPEPDIGDGCRSPGGRKRTRLYDFYVCPGHTVLT
    GCGGPREGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKGQGPCFDSSVGSGSIQGAT
    PGGRCNPLVLEFTDAGKRASWDAPKTWGLRLYRSTGADPVTLFSLTRQVLNVGPRVPIGP
    NPVITEQLPPSQPVQIMLPRPPRPPPSGAASMVPGAPPPSQQPGTGDRLLNLVEGAYQAL]
    NLTSPDKTQECWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVTSQHKLTLSEVTGQGLC
    IGAVPKTHQALCNTTQKTSDGSYYLASPAGTIWACSTGLTPCLSTTVLNLTTDYCVLVEL
    WPKVTYHSPNYVYGQFEKKTKYKREPVSLTLALLLGGLTMGGIAAGVGTGTTALVATKQF
    EQLQAAIHTDLGALEKSVSALEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFY
    ADHTGVVRDSMAKLRERLNQRQKLFESGQGWFEGLFNRSPWFTTLISTIMGPLIVLLLIL
    LFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKSIDPEEVESRE

    This is a typical Env protein, and matches other XMRV Env protein sequences present in GenBank.

    Regarding the VP62 sequence, the most current version is present at "http://www.ncbi.nlm.nih.gov/nuccore/EF185282.1".

    I don't know how the "http://www.ncbi.nlm.nih.gov/nuccore/89889045" redirect was generated, but it is incorrect. The VP62 sequence has not been discontinued. This is a GenBank error.

    I like the analogy, asleep, flex....good one! I have nightmares as well!

    Eco
     
  14. August59

    August59 Daughters High School Graduation

    Messages:
    1,480
    Likes:
    405
    Upstate SC, USA
    Is it the VP-62 version of the virus that shows very distinct preference for the human ENV as opposed to the murine ENV. This is way above my head, but could this mean that this version of an endogenus virus coul have possibly mutated or it is a variant to the initial VP62 version of XMRV. These conversations have left me more confused than before. If it has a higher preference for human ENV than murine ENV does this mean the lab workers are much vulnuerable than previously thought. Just looking for clarification in a way I can understand it and also what could possibly be the ramifications from this? Thanks to anyone that can help?
     
  15. jace

    jace Off the fence

    Messages:
    855
    Likes:
    170
    England
    They are, as you say, protein sequences you posted, Ecoclimber, not nucleotide sequences. That can be identical to say the VP62 plasmid, but it does NOT mean the nucleotide sequence is the same. That virus could be polytropic, but you won't know until the env gene in the genbank. I will give an example below.

    [​IMG]
    The nucleotide sequence is at the top, the protein at the bottom. You can translate from nucleotide to protein.

    You cannot do it the other direction for the following reason: L, for instance, can be made from nucleotide sequence ctg, ttg, cta. Each of those is different, but all translate to become L. So sequences can be very different and produce the same protein.

    You are not denying VP62 is PreXMRV-1 and of course if that were true, despite the GenBank recording the VP62 genes to have been discontinued, then the genes would morph into PreXMRV-1, and could technically be said (although I don't think that is morally right for science) to be continuing.

    It looks like the virus in 22Rv1 in the Genbank is a hypothetical virus and it has not even been shown to be xenotropic. They must have extrapolated backwards.

    The protein could be from the cell line, or it could be contamination from some unknown source.

    The important point is that the authors called this Genbank sequence the "consensus" XMRV virus, but they have not provided the nucleotide sequence for that env gene. If it is polytropic then, even though they have no evidence that a virus was created in that cell line in the 90s, who is going to be taken in that this virus is responsible for any positive result.

    It also indicates that this sequence was obtained through at least two separate process. One for the gag pol, or maybe more and one for the env. So all we have are sequences and no complete virus to go with them.

    If we only have sequences found for 22rv1 and it hasn't been shown to be replicating, they have the same issue.

    Coffin has to apply the same logic to whatever is in that cell line, which we know has at least 4 % diversity in the gag region.

    As has been pointed out before, the envelope (env) gene in the oddly named 22Rv1/CWR-R1 virus is missing. There is no env gene for that virus in the Genbank. There is /gene="gag-pro-pol", but NO /gene="env" Please try and look for yourselves.http://www.ncbi.nlm.nih.gov/nuccore/334717372

    No one has put an env gene into Genbank. Seriously, how can Coffin and Pathak have claimed that to be a "consensus" sequence?
     

See more popular forum discussions.

Share This Page