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

Discussion in 'XMRV Research and Replication Studies' started by Jemal, Jul 10, 2012.

  1. Dusty Miller

    Dusty Miller

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    What is your question? This paper finds that there are two fully-infectious endogenous retroviruses in domestic cats from Japan, and that these viruses can recombine with the well-known exogenous retroviruses of cats. An interesting report that has me looking suspiciously at my two house cats. :cat::cat: :cautious:
    Bob and Jemal like this.
  2. Mula

    Mula Senior Member

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    You posted it yourself. Prexmrv1 was not taken as a whole from any of the three sources. It was determined from all three. Unlike prexmrv2 they have not provided cloning details in the supplemental material for this 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 (GenBank accession no. FR871849)."

    I am prepared to accept this proposition for viruses which are only partial sequences and not identified. The viral fragments in 2152 are a stage before 22Rv1 but the two sections are different for this to have recombined with the other virus identified as prexmrv2. There is a section missing between the pol and env.
  3. Mula

    Mula Senior Member

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    Blomberg I believe said that NZB is a xenotropic viruses and a recombinant. Not uncommon to MLVs. I also believe NZB uses a helper virus to infect but that the helper was said to not be able to infected mouse cells.

    "XMRV, is entirely different from the likely recombination(s) which gave rise to NZB. NZB is a prototypic xenotropic virus which also seems to be a recombinant"

    https://docs.google.com/viewer?a=v&...jfdI4N&sig=AHIEtbSPZQ-aXi_rOooXCx6Ms6sM-ghIsQ
  4. Mula

    Mula Senior Member

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    Why then is there only one incomplete virus and not two individual viruses which can be compared?
  5. Bob

    Bob

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    Thanks Mula, I hadn't looked at the supplementary material before now:
    http://www.sciencemag.org/content/suppl/2011/05/31/science.1205292.DC1/1205292s.pdf

    I'm not certain, but I think that Supplemental Fig. S3. B. might provide the info that I was after.

    I'm not certain, but I think you may be misinterpreting the sentence that you've quoted, above. It looks to me like it was possible to stitch together the PreXMRV-1 genome just from the information gained either from certain xenografts, or from the HSD strain, or from the Nu/Nu mouse strains. I don't think it was necessary to have the information from all sources to define PreXMRV-1. But I can't be certain about that, until I understand it better. I don't know if it is explained in the text of the supplementary material, as I haven't read it in detail yet.
  6. ukxmrv

    ukxmrv Senior Member

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    Dr Miller,

    I am very grateful for your willingness to talk to patients here. Thank you. It is very appreciated.

    Would you be happy to help me with through this idea.

    What If by some horrible and unexpected event a mouse virus did infect humans what would be the best way to find it?

    Let's use the NZB mouse as an example.

    In New Zealand this mouse is associated with Otago medical school and as you may know there was an outbreak of M.E. in Otago (it was called the Tapanui Flu) in the mid 80's.

    One of the researchers at Otago tried using the limited technology of that time to look for a retrovirus. He (Dr Holmes) looked for the signs of RT in patients and did publish one paper.

    Given that science and methods have moved on since then what would modern researchers do if they suspected a new retrovirus has infected humans and in particular a mouse virus (like the NZB)?
  7. Dusty Miller

    Dusty Miller

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    Thank you for the encouragement to continue discussions with patients here. My experience on this site is far better than I had on the http://www.mecfsforums.com/ site! The discussion here is polite and informative, while that on the mecfsforums was hostile.

    Before I respond to your questions, would you please give me the citation for the Holmes paper that looked for signs of RT in patients? Thanks.
  8. ukxmrv

    ukxmrv Senior Member

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    Thank you for the kind and quick reply!

    Here are two of the papers by the Otago researchers. I can only find the first one as a poster presented at a conference but I'll keep looking

    1. Immunocytology and in vitro reverse-transcriptase activity in CFS
    Abstracts of Papers Presented at
    The Bi-Annual Research Conference of the
    American Association for
    Chronic Fatigue Syndrome (AACFS)

    October 10-11, 1998 -- Cambridge, Massachusetts

    Authors M.I. Holmes, R. Easingwood, J. Cross, and J. Faed

    Objective:This paper describes two blind clinical trials of paired, age, sex and ethnically matched patients with CFS; 24 pairs of patients and controls in the first, and 18 in the second. In the first, the range of duration of symptoms was 1 to 3.5 (mean 1.5) years and in the second 1 to 5 (mean 1.7) years. Peripheral blood lymphocyte (PBL) cultures were assayed in triplicate for reverse-tran scriptase (RT) activity, and examined by EM for the presence of virus-like structures at days 0 and 12 and CD2, 3, 4, 8, 16, 20, and 31, and B1 phenotypes were counted at day 0 by FACScan.
    Methods:A single dose of 1 µg ml-¹Concanavilin A (Con A) was given to all cultures at day 0. At days 4, 8 and 10 they were given 4.5 ng ml-¹ human recombinant IL-2. Cells were harvested at day 12 for EM studies, and ultracentrifuged supernatants and cells for RT assay using a poly rA:eek:ligo dT template-primer and measuring RT activity by uptake of tritiated thymidine triphosphate.

    Results:In Trial 1, RT activity up to 3 times background was observed in 9, and virus-like structures in 7 of 24 patients, and not in controls. Group means showed a significant CD4 cytopenia. In Trial 2, RT activity at levels of 2 to 4 times background were observed in 5 patients, and virus-like structures were observed in 4 of these, and not in controls. Group means showed significantly reduced CD4/CD8 ratios and an NK cytopenia. RT activity and EM virus-like structures were seen almost exclusively in the cohort of patients who identified the onset of their condition with a non-specific, acute febrile illness and whose duration of symptoms was 2 years or less.

    Conclusion: These studies suggest the in vitro RT activity and the presence of virus-like structures in PBLs may correlate in CFS with patients who relate the onset of their condition to a non-specific, acute febrile illness.

    2. Electron microscopic immunocytological profiles in chronic fatigue syndrome.

    Holmes MJ, Diack DS, Easingwood RA, Cross JP, Carlisle B.

    J Psychiatr Res. 1997 Jan-Feb;31(1):115-22.
    Source

    Department of Microbiology, University of Otago, Dunedin, New Zealand. mike.holmes@stonebow.otago.ac.nz
    Abstract

    Structures consistent in size, shape and character with various stages of a Lentivirus replicative cycle were observed by electron microscopy in 12-day peripheral-blood lymphocyte cultures from 10 of 17 Chronic Fatigue Syndrome patients and not in controls. Attempts to identify a lymphoid phenotype containing these structures by immunogold labelling failed and the results of reverse-transcriptase assay of culture supernatants were equivocal. The study was blind and case-controlled, patients being paired with age, sex and ethnically matched healthy volunteers. Prescreening of subjects included the common metabolic and immunological disorders, functional conditions and a virus-screen against hepatitis B and C, Epstein-Barr Virus, Cytomegalovirus and Human Immunodeficiency Virus.
    PMID:
    9201653
    [PubMed - indexed for MEDLINE]
  9. Dusty Miller

    Dusty Miller

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    I can't imagine why you persist in calling the 22Rv1/CWR-R1 sequence (http://www.ncbi.nlm.nih.gov/nuccore/FN692043.2) incomplete. I just did a BLAST search on this sequence, and it matches the VP62 XMRV sequence very well, with only 5 base mismatches and two single-base gaps. We know the VP62 sequence is complete because several groups, including my own, have been able to generate infectious virus from the plasmid containing the VP62 XMRV sequence. Therefore, the 22Rv1/CWR-R1 sequence also appears to be complete. The two single-base gaps in the sequence comparison are downstream of the Env coding region in both viruses, so they don't affect the protein coding regions. Both env genes encode proteins of 645 amino acids, with only one difference between the VP62 and 22Rv1/CWR-R1 Env amino acid sequences (top sequence = VP62, middle sequence = shared amino acids, bottom = 22Rv1/CWR-R1 sequence):

    ...Query 1 MESPAFSKPLKDKINPWGPLIIMGILVRAGASVQRDSPHQVFNVTWKITNLMTGQTANAT 60
    ...........MESPAFSKPLKDKINPWGPLIIMGILVRAGASVQRDSPHQVFNVTWKITNLMTGQTANAT
    Sbjct 5754 MESPAFSKPLKDKINPWGPLIIMGILVRAGASVQRDSPHQVFNVTWKITNLMTGQTANAT 5933

    ..Query 61 SLLGTMTDTFPKLYFDLCDLVGDNWDDPEPDIGDGCRSPGGRKRTRLYDFYVCPGHTVLT 120
    ...........SLLGTMTDTFPKLYFDLCDLVGDNWDDPEPDIGDGCRSPGGRKRTRLYDFYVCPGHTVLT
    Sbjct 5934 SLLGTMTDTFPKLYFDLCDLVGDNWDDPEPDIGDGCRSPGGRKRTRLYDFYVCPGHTVLT 6113

    .Query 121 GCGGPREGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKGQGPCFDSSVGSGSIQGAT 180
    ...........GCGGPREGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKGQGPCFDSSVGSGSIQGAT
    Sbjct 6114 GCGGPREGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKGQGPCFDSSVGSGSIQGAT 6293

    .Query 181 PGGRCNPLVLEFTDAGKRASWDAPKTWGLRLYRSTGADPVTLFSLTRQVLNVGPRVPIGP 240
    ...........PGGRCNPLVLEFTDAGKRASWDAPKTWGLRLYRSTGADPVTLFSLTRQVLNVGPRVPIGP
    Sbjct 6294 PGGRCNPLVLEFTDAGKRASWDAPKTWGLRLYRSTGADPVTLFSLTRQVLNVGPRVPIGP 6473

    .Query 241 NPVITEQLPPSQPVQIMLPRPPRPPPSGAASMVPGAPPPSQQPGTGDRLLNLVEGAYQAL 300
    ...........NPVITEQLPPSQPVQIMLPRPPRPPPSGAASMVPGAPPPSQQPGTGDRLLNLVEGAYQAL
    Sbjct 6474 NPVITEQLPPSQPVQIMLPRPPRPPPSGAASMVPGAPPPSQQPGTGDRLLNLVEGAYQAL 6653

    .Query 301 NLTSPDKTQECWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVTSQHKLTLSEVTGQGLC 360
    ...........NLTSPDKTQECWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVTSQHKLTLSEVTGQGLC
    Sbjct 6654 NLTSPDKTQECWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVTSQHKLTLSEVTGQGLC 6833

    .Query 361 IGAVPKTHQALCNTTQKTSDGSYYLASPAGTIWACSTGLTPCLSTTVLNLTTDYCVLVEL 420
    ...........IGAVPKTHQALCNTTQKTSDGSYYLASPAGTIWACSTGLTPCLSTTVLNLTTDYCVLVEL
    Sbjct 6834 IGAVPKTHQALCNTTQKTSDGSYYLASPAGTIWACSTGLTPCLSTTVLNLTTDYCVLVEL 7013

    .Query 421 WPKVTYHSPNYVYGQFEKKTKYKREPVSLTLALLLGGLTMGGIAAGVGTGTTALVATKQF 480
    ...........WPKVTYHSPNYVYGQFEKKTKYKREPVSLTLALLLGGLTMGGIAAGVGTGTTALVATKQF
    Sbjct 7014 WPKVTYHSPNYVYGQFEKKTKYKREPVSLTLALLLGGLTMGGIAAGVGTGTTALVATKQF 7193

    .Query 481 EQLQAAIHTDLGALEKSVSALEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFY 540
    ...........EQLQAAIHTDLGALEKSVSALEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFY
    Sbjct 7194 EQLQAAIHTDLGALEKSVSALEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFY 7373

    .Query 541 ADHTGVVRDSMAKLRERLNQRQKLFESRQGWFEGLFNRSPWFTTLISTIMGPLIVLLLIL 600
    ...........ADHTGVVRDSMAKLRERLNQRQKLFES QGWFEGLFNRSPWFTTLISTIMGPLIVLLLIL
    Sbjct 7374 ADHTGVVRDSMAKLRERLNQRQKLFESGQGWFEGLFNRSPWFTTLISTIMGPLIVLLLIL 7553

    .Query 601 LFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKSIDPEEVESRE 645
    ...........LFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKSIDPEEVESRE
    Sbjct 7554 LFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKSIDPEEVESRE 7688

    As I stated earlier in this thread, the fact that the env gene is not annotated in the 22Rv1/CWR-R1 sequence doesn't mean that it is not there. Analysis of the GenBank sequence shows clearly that it is present.

    Regarding your question of why separate sequences for the 22Rv1 and CWR-R1 viruses are not provided, I believe the authors of the study found that the CWR-R1 sequence matched that of the 22Rv1 sequence exactly, and because the two viruses represent descendants of the same original recombinant virus, they decided to report only one sequence. Why report two sequences when they are identical and derive from the same parental virus?

    If you are still confused, please tell me exactly what bothers you.
  10. anciendaze

    anciendaze Senior Member

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    An initial opinion was all I asked for.
    BTW: I'm sure your cats return that look. I often wonder where cats get their secret information.

    I did have a comment on the example of the "harmless" ERV in NZB mice. Those mice were carefully selected for health. Other lines formed from the same basic NZ genetic stock are used as laboratory models of a number of diseases, including chronic lymphocytic leukemia, lupus, other autoimmune disorders and neurodevelopmental pathology. Even a lesser degree of selection would allow me to argue that polio virus is harmless to properly-selected humans.
  11. Dusty Miller

    Dusty Miller

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    Perhaps you know, I haven't had time to look. Do the NZB mice have a functional Xpr1 receptor for xenotropic viruses, such as the NZB virus? If not, is it hypothesized that the NZB virus causes disease without being able to infect the cells of the NZB mouse?
  12. anciendaze

    anciendaze Senior Member

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    I don't yet know if they do, though I have been assuming not. Even if databases say one thing, I feel sure nobody has checked all the strains in detail. This might be the difference between health and illness. I was only aware NZB mice were once considered as an animal model in geriatric psychiatry, which caught my eye as possibly related to this damned illness. Patients on this forum will find the description in the abstract evocative. I was just about to add the link to the previous post when I saw your response.
  13. jace

    jace Off the fence

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    That's the extrapolation from the protein sequence you posted, Dr. Miller. Like I said, many viruses share proteins. It's not the env gene. There is no env gene in Genbank, even after updating.

  14. Dusty Miller

    Dusty Miller

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    There clearly IS an env gene in the 22Rv1/CWR-R1 sequence posted in GenBank. A typical gene consists of a transcriptional promoter (including sequences that regulate transcription), an open reading frame downstream of the promoter that encodes a protein, and signals that terminate transcription downstream of the open reading frame. Splice signals can be present in the gene that lead to removal of certain sections of the RNA during processing to make the mature messenger RNA. Are we OK so far? The env gene in the 22Rv1/CWR-R1 XMRV sequence consists of the transcriptional promoter in the long terminal repeat (LTR) present at the beginning of the sequence, the open reading frame for the Env protein which runs from a translation start codon to a stop codon present at bases 5754..7691, followed by signals that terminate transcription and lead to addition of a polyA tail to the RNA in the LTR at the end of the sequence. There are splice signals in this long transcript that lead to removal of sequences from just after the left LTR to just before the start codon of the open reading frame that encodes the Env protein. It is this spliced region, or intron, that is not annotated in the 22Rv1/CWR-R1 GenBank file, but this splice is typical of retroviruses. Partial splicing of this intron allows production of the Gag and Gag-Pol proteins from a full-length unspliced RNA and production of the Env protein from the spliced RNA.

    Jace, what more do you need to convince you that there is an env gene? Can someone else help me out here and tell me what Jace has in mind that I am missing? Jace, if you had your whole genome sequenced, and they gave you the complete 6 billion base sequence, would you complain that no genes were present because they were not annotated?
    Sam Carter likes this.
  15. jace

    jace Off the fence

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    Dr. Miller, you posted yourself the quote from Genbank that says
    pu·ta·tive (py[​IMG][​IMG]t[​IMG]-t[​IMG]v)​
    adj.
    Generally regarded as such; supposed. See Synonyms at supposed.​
    [Middle English, from Old French putatif, from Late Latin put[​IMG]t[​IMG]vus, from Latin put[​IMG]re, to prune, think; see pau-2 in Indo-European roots.]
    You have also posted what is the sequence for a protein, extrapolated from the protein linking it to the env of 'XMRV' more specifically VP62. If they had the env sequence, surely they would have posted the env sequence? Without that, there is no way we can be sure that this is VP62.
  16. free at last

    free at last Senior Member

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    Hi Mr Miller. I have no expetise to add here. But i would like to say thank you very much for trying to help the community. Just thinking through the problems. and learning about this horrible illness ( possibly illnesses ? ) is so much appreciated. we really need you more than you can ever imagine. I recovered to maybe 80 90% of my health. Though in the begining it was horrible. really frightening symptoms. Very much like influenza. temps of 102 or higher. hot and cold shivering. aching. you get the picture. I cried when symptoms started ( im supposed to be a man ) because i just couldnt take the claustrophobic symptoms at all. And it was always again and again. it always came back ? My story is quite interesting. i belive i have a lot to say. the picture can be enlightening. when you learn how the illness started and progressed to the present day. like for example the long gaps of flu like attacks with high temps. then similair symptoms now much more frequent ( every week ) Temps gradually becoming more under control. A virus that no one else in my family caught ? but one that obviously was dormant. that could keep sufacing. that my body was trying to learn to live with. ( which it has. does ? ) I belived xmrv was the answer. I tested postive 3 out of the tests done on the uk 50 wpi study. But now we know all is not what it seems. But its a virus Dr Miller. in my heart i know its a damm virus. And or immune dysfuntion.Hope you keep trying to help people that are as ill as i was. they need you more than you know. Thank you so much for becoming interested
    Neil
  17. anciendaze

    anciendaze Senior Member

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    It may be useful to narrow questions about the XMRV envelope to the functional immunosuppressive domain found by Schliect-Louf/Heidmann and also the subject of a patent.

    Should such a domain be inserted in the envelope of a common virus it could have significant public health implications. This is not some wild speculation, as entire working retroviral genomes have been found inserted in fowlpox virus and fragments in viruses of the herpes group. Transmission of an altered virus, or viral envelope, to humans is more likely if most carriers are asymptomatic, and people commonly eat the original host species. Marek's Disease Virus has considerable homology to some human herpes viruses.

    Has anyone looked for that domain elsewhere, or is the whole issue too far fetched for experts to even consider?
  18. jace

    jace Off the fence

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    Hey Free, I hear ya. I was one of the Ashford 50 too, and we have that and the positive result in common. I know it's not "XMRV VP62" that's making us ill, it's likely some kind of gamma retrovirus though. Anyone read Anciendaze' excellent blog series on the subject?

    My take on all the stuff that gets posted by people trying to discredit retroviral research in pwME is similar to Shakespeare's quote from Hamlet - "The(y) lady doth protest too much, methinks".

    I reckon this virus is pretty wimpy though. I may well be winning too, who knows, with a combo of endocrine supplements and herbals.

    The very ferocity of the negative campaign rings alarm bells with me.
  19. currer

    currer Senior Member

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    We know from JT1024's post on the last page that there IS concern about retroviral transmission into the population from biologicals - but this genuine concern is NEVER admitted to in public in the debate over ME/CFS or "XMRV".
    It is this evasion which creates mistrust and suspicion from patients.

    There is another good hypothesis for ME which is that it could be an auto-immune syndrome provoked by vaccine adjuvants.
    http://www.ncbi.nlm.nih.gov/pubmed/20708902
    http://www.ncbi.nlm.nih.gov/pubmed/21776441
    http://www.ncbi.nlm.nih.gov/pubmed/21051205
    About 5% of CFS/ME cases commence immediately following a vaccination. But this can never be discussed either.
    I was present at a talk at the London School of Hygeine and Tropical Medicine when this question was asked - and none of the four doctors on the panel would respond. In fact they behaved as if they did not hear the question.

    Now is this a healthy situation for a society as dependent on vaccination and science as our own?
    Is it wrong to suggest that some people become ill following vaccination?
  20. Bob

    Bob

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    As you probably know, Jace, I've always rigorously defended XMRV research. And I have often agreed with what you've said above, especially as many people have often appeared to be prematurely very motivated to discredit all research into XMRV, especially in relation to ME. But this thread has been very civilised, with people only expressing honest opinions, based on their understanding of the science.

    I've got to the point now where I find it very difficult to believe that there are any retroviruses involved in ME. It's been at least two years now since the Lombardi paper, and there haven't been any convincing positive retroviral studies into ME. But there must have been loads of scientists attempting to replicate the research, or trying to find XMRV-related viruses, but unable to. Negative studies often don't get published, and just get abandoned, so we probably aren't even aware of many of them.

    Also, Judy has mentioned contamination on a number of occasions now. The latest media article is just one of those occasions. Obviously, contamination is an issue, and could explain many of the positive results. I know it doesn't explain everything, but neither have there been any other convincing explanations (that have been backed up by convincing research results) for the various positive MLV results.

    I'm still fascinated by retroviral research, and I'm looking forward to watching it continue. I think retroviruses could play a role in other diseases, such as breast cancer. But with ME, I'm not convinced, and I'm looking elsewhere for research developments now, such as Rituximab, and the ongoing biomarker research, and Lipkin's CFI pathogen study which I hope could be ground-breaking.

    Also, we've been promised specific results of next generation sequencing from the WPI, and from Dr Snyderman, for months now, or years, and the results have not come to light. So I can only draw one conclusion from that, and that's that they did not confirm their expectations.

    So, it appears that: next generation sequencing has not confirmed the other research results; there has been contamination; there have been no confirmation studies for a human virus in ME; and Judy has not released any further information regarding her other retroviruses in two years, but has now discussed contamination issues in public.

    If anyone wants to remind me if I'm missing anything that would convince me here, then I'm open minded, and receptive.

    I also think it's important to remember that many retrovirologists around the world will probably have been attempting to replicate the XMRV research and will have been researching the newly discovered MLVs, such as with the recent PreXMRV-2 paper. Research into MLVs has probably accelerated since the discovery of XMRV, with loads of resources available. There are going to be many scientists around the world who are extremely interested and motivated in finding out more about: newly discovered MLVs; potential new human viruses; the contamination of cell lines; and the various MLVs that are so ubiquitous.

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