Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Naïve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

<div><h3>Background</h3><p>Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.</p> <h3>Methodology/Principal Findings</h3><p>DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV <em>gag</em> and <em>env</em> and one sensitive, published nested PCR assay targeting <em>env</em>. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for <em>gag</em> by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.</p> <h3>Conclusions/Significance</h3><p>The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.</p> </div

Summary of XMRV screening results.

a<p>Fractions are: number of subjects scoring positive/total number of subjects screened.</p>b<p>Ab, antibody.</p

Screening for XMRV in patient PBMCs by PCR.

<p>PCR products were analyzed on agarose gels containing ethidium bromide. (A) Representative gels for non-nested <i>env</i> (top panel) and non-nested <i>gag</i> (bottom panel) PCRs are shown containing a set of three replicates for each of 5 HIV-1⁺ patient samples. A yellow arrow indicates the sole PCR band, from patient 103219, found to be comprised of XMRV DNA by sequencing. (B) A representative gel for nested <i>env</i> PCR is shown for the same 5 HIV-1⁺ patient samples depicted in (A). Vertical black arrows in (A) and (B) indicate lanes from patient 103219 containing either (A, bottom panel) a band comprised of XMRV sequence or (B) a band of the expected mobility for the target sequence. (m) 100 base pair molecular weight marker, (1∶10⁴) DNA from one infected cell diluted in DNA from 10⁴ uninfected cells used as template for positive control.</p

Detecting murine DNA by IAP PCR.

<p>PCR products were analyzed on 1.5% agarose gels containing ethidium bromide. (A) Sensitivity of the IAP PCR assay was determined by performing PCRs on titrations of EL4 murine cell line DNA in a background of 200 ng LNCaP DNA. One murine cell equivalent (1 eq) indicates 6 pg of EL4 DNA. XMRV-infected LNCaP (iLNCaP) and uninfected LNCaP (uLNCaP) were included as controls. (B) Screening results for 17 HIV-1⁺ patient samples. Arrow points to sample 103219, which tested positive for XMRV by non-nested gag PCR. (m) 100 base pair molecular weight marker, (EL4) 6 pg of murine EL4 cell line DNA without a background of human DNA.</p

Primers used for screening PBMC DNA specimens for XMRV.

a<p>Location of 5′ end of forward primer target site to 3′ end of reverse primer target site on XMRV VP62 reference genome (accession no. DQ399707.1).</p

Sensitivity analysis of XMRV PCR assays.

<p>PCR products were analyzed on agarose gels containing ethidium bromide. (A) Non-nested PCR assays targeting the XMRV <i>env</i> gene (top panel) and the <i>gag</i> gene (bottom panel), and (B) a nested PCR assay targeting the XMRV <i>env</i> gene were evaluated for their ability to detect either (A) provirus in XMRV-infected PNT1A cell DNA or (B) provirus in XMRV-infected LNCaP cell DNA diluted in uninfected cell DNA. Dilutions of infected cells in uninfected cells are indicated by ratios, i.e. 1∶10⁴ indicates one infected cell diluted in 10⁴ uninfected cells. (m) 100 base pair molecular weight marker, (H₂O) water used in place of DNA template as a negative control, (u) uninfected PNT1A DNA used as template for negative control.</p