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Lack of the Detection of XMRV or Polytropic MLV-Related Sequences in Blood Cells From HIV-1–Infected Patients in Spain

Luczkowiak, Joanna MSc*; Martínez-Prats, Lorena MSc*; Sierra, Olalla CLT*; Fiorante, Silvana MD; Rubio, Rafael MD; Pulido, Federico MD; Otero, Joaquin R. MD*; Delgado, Rafael MD*

JAIDS Journal of Acquired Immune Deficiency Syndromes: 1 February 2012 - Volume 59 - Issue 2 - p 101–104
doi: 10.1097/QAI.0b013e318238b596
Basic and Translational Science

Background: Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)–related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus.

Methods: Forty HIV-1–infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination.

Results: No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed.

Conclusions: XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.

*Laboratory of Molecular Microbiology, Instituto de Investigación i+12, Hospital Universitario 12 de Octubre, Madrid, Spain

HIV Unit, Instituto de Investigación i+12, Hospital Universitario 12 de Octubre, Madrid, Spain

Correspondence to: Rafael Delgado, MD, Laboratory of Molecular Microbiology, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041 Madrid, Spain (e-mail:

Supported by funds from EU-FP7 (PITN-GA-2008-213592, CARMUSYS), ISCIII-FIS080806, and FIPSE-36749 to R.D.

Partially presented at 18th Conference on Retroviruses and Opportunistic Infections, February 27th to March 2, 2011, Boston, MA. Abstract #236.

The authors have no conflicts of interest to disclose.

Received July 29, 2011

Accepted September 20, 2011

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Xenotropic murine leukemia virus–related virus (XMRV) was described in 2006 by Urisman et al1 in prostate cancer stromal cells. XMRV was considered the first gammaretrovirus infecting humans.2 The sequence of XMRV is closely related to murine leukemia virus (MLV): the homology range between 66% and 96% (envelope and protease sequences, respectively).1 The newly described infectious agent can infect foreign cells, including humans, but cannot reinfect murine cells. This property distinguishes xenotropic viruses from ecotropic MLV that can infect only murine cells or polytropic MLV that infects both murine and foreign cells.3

In 2009 Lombardi et al4 reported a strong association between XMRV and chronic fatigue syndrome (CFS). XMRV DNA was detected in blood cells from 67% of patients with CFS as compared with 3.7% detected in healthy blood donors. This study raised important controversy because a number of subsequent studies could not detect any proviral DNA sequences in patients with CFS.5–8 However, Lo et al9 reported the presence of MLV-related sequences in 86.5% of patients with CSF and 6.8% of healthy controls. The sequences detected in this study were slightly different than the XMRV sequences previously described. The virus detected in patients was closer to polytropic MLV viruses infecting both murine and human cells.9 The fact that XMRV was detectable in a small but significant proportion of healthy blood donors raised concern about potential transmission by blood transfusion, and health authorities in Canada and United States decided to exclude patients with CFS from blood donation.10 Nevertheless XMRV or polytropic MLV remained undetectable by many different laboratories in different clinical conditions and control groups.11–14

As the existence of XMRV raises many questions, including its mode of transmission, we decided to study the presence of XMRV or polytropic MLV-related sequences in a group of HIV-1–infected patients with a high proportion of intravenous drug users (IDUs) and coinfection with hepatitis C virus (HCV) because this group is associated with a higher risk of infection with other potential blood-borne agents.

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Whole blood samples used in this study were obtained from 40 HIV-1–infected patients upon informed consent under an internal review board approved protocol. All patients were on antiretroviral therapy (ART), and 27 of 40 (67.5 %) had undetectable HIV-1 viral load (<50 copies of HIV-1 RNA/mL) in plasma. Combination regimes included drugs with in vitro activity against XMRV (zidovudine, tenofovir, or raltegravir )15 in 31 of 40 (77.5 %). Within the group of patients, 23 (57.5 %) were men and 20(50 %) were active or former IDU. Additionally, 24 (60 %) of patients were coinfected with HCV [19 of 20 (95 %) of IDU were HCV+] and 22 (55 %) were coinfected with Hepatitis B virus.

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DNA Isolation and Polymerase Chain Reaction

DNA was extracted from 400 μL EDTA-whole blood according to manufacturer's instructions (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany). Each sample was amplified by nested polymerase chain reaction (PCR) using 2 sets of primers targeted to gag XMRV-specific sequence (first PCR: 419F/1154R; second PCR: GAG-I-F/GAG-I-R or NP116/NP117) and 1 set of primers corresponding to env region of XMRV (first PCR: 4672F/7590R; second PCR: 5922F/6273R).4,9 Primers for human beta-globin (hβG) (hβG-FO-148 /hβG-RO-296) were used as a control for DNA amplification.5 Negative controls in the absence of DNA were included in each experiment.

Each first-round PCR reaction was performed in a final volume of 50 μL using 500–650 ng of total cellular DNA for reaction. For both, gag and env, PCR reaction conditions were used as previously described.4,9 To exclude the possibility of potential contamination of the samples used in the study by murine DNA, seminested PCR for the detection of mouse mitochondrial DNA (mt DNA) was performed. The external PCR primers were as follows: mt15982F/mt16267R and internal primers were as follows: mt16115F/mt16267R.9 PCR conditions were the same as for gag and env nested PCR.

To evaluate PCR sensitivity the full-length molecular viral clone Vp62-XMRV (obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD)] from R. H. Silverman and B. Dong) was used in each batch of PCR reactions as a positive XMRV control.

PCR amplicons were visualized on a 2% agarose gel stained with ethidium bromide. PCR products from the first-round and second-round PCR of the correct size were excised from the gel and purified using Mini Elute Gel Extraction Kit (Qiagen). PCR products were sequenced by Big Dye chemistry using primers corresponding to gag region of XMRV (NP116/NP117) and analyzed by VectorNTIv11 software.

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We tested DNA extracted from whole blood samples from 40 HIV-1–infected patients. For each patient, the single-round PCR for hβG and gp120 V3 were positive confirming a reliable and sensitive DNA amplification. Precise sensitivity of proviral DNA detection was estimated by amplification of the gag region of the control plasmid XMRV-Vp62 in ≤10 copies. We could not detect any XMRV or polytropic MLV-related virus sequences, either by the gag or env PCR, in any sample from patients or controls (95% confidence interval: 0% to 8.8%). In 4 of 40 patients, PCR targeting gag yielded products of the expected size (Fig. 1). Sequencing and BLAST ( analysis of these amplicons showed >99 % sequence identity to a mouse endogenous retrovirus in patients 1–3 (respectively, Mus musculus chromosome 1, Mus musculus chromosome 12, Mus musculus chromosome 11) and 100 % identity to XMRV in patient 4 (98% of identity to Mus musculus chromosome 12). These positive results could not be confirmed in subsequent blood samples from the same 4 patients. Interestingly, the initially positive 4 samples were also positive for mouse mtDNA by PCR, whereas a negative result was obtained in 15 samples tested of the remaining 36 patients that were negative for XMRV. The nested PCR for gag and env region of XMRV or polytropic MLV-related viruses described above yielded a rather complex band pattern visible on the gel.9,13 However, exhaustive sequencing of those bands and subsequent analysis by BLAST systemically revealed a 100% identity with human chromosome DNA.

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XMRV or MLV-related sequences were not amplified from DNA obtained from blood cells of HIV-1–infected patients in Spain. We were not able to detect any specific viral sequence by nested PCR with primers corresponding either to gag or env genes of XMRV/MLV. Consistently with some other studies, our results although limited by the number of patients reasonably excludes a significant prevalence (95% confidence interval: <10%) of XMRV/MLV-related virus among HIV-1–infected patients with a high proportion of IDU and coinfection with HCV. The failure to detect MLV-related sequences in our group of patients is unlikely due to the methods used in the study, such as isolation and amplification of XMRV DNA, because both hβG and the V3 region of HIV-1 envelope were successfully amplified in all patients. XMRV or MLV-related virus amplification strategy used in our study was performed with DNA input necessary for reliable PCR results (600 ng) and with identical primer sets that were used in other published methods able to detect virus DNA in patients.4,5,9

Most of our patients were on ART including at least 1 drug reported to be active in vitro against XMRV: raltegravir, tenofovir and/or zidovudine,15 however, because our diagnostic procedures targeted cell-associated DNA including integrated proviruses and based in our knowledge of the stability of detection of HIV-1–infected cells during treatment,16 it is unlikely that ART could have contributed to a significant decrease in the number of cells with integrated XMRV yielding thus negative results using a highly sensitive nested PCR technique. It is also worth noting that although most of our patients on ART had undetectable HIV-1 viral load in plasma, we were able to detect, using similar procedures, a fragment corresponding to the sequence of HIV-1 envelope from blood cells in all cases.

Several factors might be responsible for negative findings on XMRV/MLV-related virus infection, such as geographic distribution, possible genetic variations of the virus, patients' selection. Geographical distribution may play an important role because most of the studies detecting specific XMRV sequences were performed on patients from North America.1,2,4,9 It might reflect the differences between American and European populations. Genetic differences in viral strains might be also considered as a possible reason for different virus detection. However, it seems unlikely due to the high degree of XMRV sequences conservation among strains isolated from different sources.17 Related to this fact, it is of great importance the recent report by Paprotka et al18 showing that XMRV is most likely the result of a recombination of 2 endogenous murine retroviruses during in vitro passage of a human prostate tumor in a nude mice model.

On the other hand, our results in accord with other studies have raised a concern about contamination with murine DNA that could account for the positive result in gel electrophoresis of PCR products. This risk of contamination might be due to the frequent usage of mice in biomedical research laboratories worldwide. Each mouse cell contains in the DNA more than 100 MLV genomes (endogenous viruses), which are the result of past infections of mouse germ cells and subsequent integration into the germline.19 Most of those endogenous viruses are defective and cannot lead to productive virus infection. However, PCR primers specific for the detection o XMRV/MLV-related sequences might often amplify those endogenous viral sequences giving “false positive” results.20 To exclude the possible contamination of the samples, Lo et al9 described the seminested PCR strategy for the detection of mouse mitochondrial DNA that should be used during XMRV screening. Many different possible sources of contamination with murine DNA have been previously described. It has been shown that heparin, knives for slicing tumours, monoclonal antibodies, Hot Start Taq polymerase, enzymes mixtures of 1-step reverse transcriptase—polymerase chain reaction, fetal calf serum, or phosphate-buffered saline might be contaminated with endogenous mouse retroviral DNA.3,13,21–24

In our study, we obtained mouse endogenous retroviral sequence amplification from 3 samples and XMRV in one, however, we considered this was most likely due to cross contamination because the presence of murine DNA was confirmed exclusively in these samples, and we could not replicate these findings from newly extracted samples of those 4 patients. The detection of a sequence identical to XMRV in one sample with demonstrated contamination by murine DNA could be the result of cross contamination with the control plasmid XMRV-Vp62 because it was not confirmed in an additional sample from the same patient.

In summary, we demonstrated no evidence of XMRV/MLV-related virus infection in a group of patients infected with HIV-1 in Spain with a high degree of blood-borne pathogen exposition. This makes highly unlikely the possibility that XMRV is being transmitted by blood products. Importantly, our results suggest that some of the positive results previously reported by other groups could be associated to murine DNA or XMRV vectors contamination in samples or reagents.

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blood-borne agents; chronic fatigue syndrome; Hepatitis C virus; HIV-1; XMRV

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