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Partial molecular cloning of the JHK retrovirus using gammaretrovirus consensus PCR primers

Discussion in 'Other Health News and Research' started by Ecoclimber, Apr 3, 2014.

  1. Ecoclimber

    Ecoclimber Senior Member

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    I originally posted this thread in the apparent 'dead' xmrv archives forum. Just wanted people to be aware of this thread

    http://forums.phoenixrising.me/inde...irus-consensus-pcr-primers.29396/#post-448795

    Future Virol. 2013 May;8(5):507-520.
    Partial molecular cloning of the JHK retrovirus using gammaretrovirus consensus PCR primers.
    Halligan BD1, Sun HY, Kushnaryov VM, Grossberg SE.
    Author information
    Abstract

    The JHK virus (JHKV) was previously described as a type C retrovirus that has some distinctive ultrastructural features and replicates constitutively in a human B-lymphoblastoid cell line, JHK-3.

    In order to facilitate the cloning of sequences from JHKV, a series of partially degenerate consensus retroviral PCR primers were created by a data-driven design approach based on an alignment of 14 diverse gammaretroviral genomes. These primers were used in the PCR amplification of purified JHK virion cDNA, and ana lysis of the resulting amplified sequence indicates that the JHKV is in the murine leukemia virus (MLV) family. The JHK sequence is nearly identical to the corresponding region of the Bxv-1 endogenous mouse retrovirus (GenBank accession AC115959) and distinct from XMRV. JHKV gag-specific amplification was demonstrated with nucleic acids from uncultivated, frozen, peripheral blood mononuclear cells (PBMCs) of the index patient, but not in PBMCs from nine healthy blood donors.

    Unlike earlier reports, in which MLV-like sequences were identified in human source material, which may have been due to murine contamination, budding retrovirions were demonstrated repeatedly by electron microscopy in uncultivated lymphocytes of the index patient that were morphologically identical in their development to the virions in the JHK-3 cells, and immunological evidence was obtained that the index patient produced IgG antibodies that bound to the budding viral particles in patient PBMCs and in the JHK-3 cells.

    These data indicate that the patient had been infected by JHKV, lending significance to the demonstration of JHKV amplicons in nucleic acids of the patient's PBMCs. In future studies, the PCR primer sets described herein may expand the detection of an amplifiable subset of viruses related to MLV.

    KEYWORDS:
    B-lymphoblastoid cells, Epstein–Barr virus, electron microscopy, gammaretrovirus, murine leukemia virus
    Click on Link Above Full Article Access

    ....Establishing a possible etiologic relationship would require a rather large clinical study of patients and controls. Although the history of the patient’s ill-defined, viral-like illness, from which the patient recovered completely some years later, bears a resemblance to CFS/ME (among other disease complexes), no definite diagnostic marker yet exists for CFS/ME.....

    ....No study to our knowledge has shown a direct relationship to viral particles infecting the patient, such as we have shown by the ultrastructural similarity of the budding viral forms in the uncultivated PBMCs of IP to those in the JHK-3 cells.

    ....Based on the evidence presented, we do not conclude that the JHK virus is a ‘human retrovirus’. Whether the JHK virions observed, the amplicons sequenced or possibly JHKV-related gammaretroviruses may be involved in identifiable human infection awaits further study, for which our PCR primers may enable detection.

    Eco Note:
    Grossberg has been researching JHKV, Epstein–Barr virus (EBV)-positive human B-cell line for more than a decade. However, on one of his research papers, he stated that the JHK-3 retrovirus in his findings was closely associate with the xmrv sequences in GenBank. It was not and was asked to remove the association which he did.

    This was on JHK-3 retrovirus 5 LTR, partial sequence; and (gag) gene, partial cds GenBank HM119591.1 DNA Seuquence.
     
    Last edited: Apr 4, 2014
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  2. Bob

    Bob

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    England (south coast)
    Thanks Eco. I spotted it in the XMRV archive, but I'm set up to get alerts for XMRV forums. We do seem to get alerts to the XMRV archive. (I'm not quite sure why all XMRV sub-forums have been placed in an archive.)

    If anyone is interesting in keeping up with XMRV news, then I suggest 'watching' the XMRV research sub-forum, which we still post in from time to time:
    http://forums.phoenixrising.me/index.php?forums/xmrv-research-and-replication-studies.27/
     
    Last edited: Apr 3, 2014
  3. natasa778

    natasa778 Senior Member

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    Oh no, an MLV-related, C-type retrovirus INFECTING a human and living it large in hisB cells?!?

    Someone better debunk this study quick and make it go away, in case some silly people get some silly ideas.
     
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  4. natasa778

    natasa778 Senior Member

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  5. asleep

    asleep Senior Member

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    Just posted this in the other thread on this study...didn't realize there were two.

    One of the more interesting quotes from the paper, made in reference the "final word" study by Lipkin (Alter et al):
    In other words, they could not have detected JHKV in that study. Therefore, as people said at the time, the Lipkin study wasn't nearly as "final" as it was publicized to be with regards to MLV-related viruses.
     
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  6. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    Gotta ask why Lipkin didn't bother to look for JHKV.
     
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  7. anciendaze

    anciendaze Senior Member

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    They still don't have a full sequence for JHKV. If you read this paper carefully you will notice that this team used a degenerate primer set designed for highly-conserved regions of gamma retroviruses. This means they could pull up quite a number of different retroviruses.

    A second problem is recombination with HERVs. While this might be rare in cases of acute infectious disease, in cases of chronic disease lasting a number of years it is more likely to be the rule than the exception. If you do not know the particular sequence, and are rejecting things that look like HERV sequences, it is easy to overlook a slow infection hiding in the underbrush created by transcription of HERVs.

    (Recombination between different sequences causes serious problems in HIV research. I doubt whatever cause is ultimately found for ME/CFS will remain consistent at the level of particular sequences anymore than HIV infection does. The extreme limits of variation in HIV sequences show only 50% homology with other HIV sequences. I'm not sure what the range of variation is in individual patients, and I'm not sure the experts know. If they did they might be able to eradicate the active disease.)

    I notice that this index patient is said to have fully recovered after a number of years. This might be merely a subjective impression, as Dr. Bell found in his ME/CFS patients. On the other hand it could also reflect convergence of active strains of JHKV with HERVs the patient's immune system was able to hold latent.

    Aside: while reading this, I noticed that one test for active gamma retroviruses involved looking for manganese-dependent reverse transcription. As a curious example of a possible coincidence, I recall that borrelia burghdorferi was recently discovered to use manganese where other bacteria use iron. This defeats defensive measures which try to keep iron ions out of infected tissues. How common is this strategy in pathogens causing chronic disease?
     
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