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

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.
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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

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....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.

 

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.