No evidence of xenotropic murine leukaemia virus-related virus in French blood donors

Dear Sir,

Xenotropic murine leukemia virus-related virus (XMRV) is a Gammaretrovirus, family Retroviridae, identified initially in 2006, and closely related (>95% nucleotide identity) to murine leukaemia viruses. This enveloped virus possesses a genome consisting in a dimer of linear, single-stranded RNA, approximately 8,200 nucleotides long. The prototype of the viral sequence contains three major open reading frames, i.e. gag (536 amino acids), pol (1,197 amino acids) and env (645 amino acids), flanked by two long terminal repeat sequences.

XMRV sequences were first detected in human prostate tumours and later identified in the blood of patients with chronic fatigue syndrome¹. These findings motivated subsequent and conflicting epidemiological investigations, with a few studies revealing a high prevalence of XMRV (up to 86%) while many others were unable to detect the virus in various different cohorts of humans. Some studies suggested that the prevalence of the virus (viral RNA or proviral DNA form) in healthy subject is not negligible (∼3% to ∼7%), raising concerns about its potential transmission by blood transfusion². Of note, most of these studies involved relatively small cohorts.

We investigated the presence of XMRV RNA in 671 blood samples from healthy blood donors using two distinct real-time assays. The blood donors (300 women and 371 men, mean age 40 years, male-to-female sex ratio 1.23) lived in south-eastern France. Blood samples were collected in vacuum tubes (Vacutainer, SST, Becton Dickinson, Meylan, France), centrifuged, and 1 mL plasma aliquots were stored at −80 °C until use. Nucleic acids were extracted from the 1 mL plasma volumes (MagNA pure LC, Roche Diagnostics, Meylan, France) and tested for XMRV genomic RNA by real-time TaqMan reverse transcriptase polymerase chain reaction (RT-PCR; StepOne Plus, Applied Biosystems, Courtaboeuf, France) immediately after extraction in order to avoid freezing/thawing cycles. Amplification reactions were performed using one tenth of the eluted material with the AgPath-ID One-Step RTPCR kit (Applied Biosystems) in a final volume of 25 μL. Two detection systems were tested in separate amplification assays, with target sequences located either on the gag gene (Gag-3S/Gag-3A primers, Gap p probe, 83 nt) or on the pol gene (XMRV4552F/XMRV4653R/XMRV4673R primers, XMRV4572 probe, 121 nt) of the viral genome^3,4. Both probes were labelled with 5′-FAM and 3′-TAMRA. Amplification conditions were 45 °C for 10 min and 95 °C for 10 min, followed by 60 cycles of 95 °C for 15 s and 60 °C for 45 s. Using dilutions of two specific synthetic templates, we estimated the sensitivity of both TaqMan assays to be 20 copies of XMRV target sequence. A murine leukaemia virus contamination control (mouse mitochondrial DNA PCR)⁵ performed on the real-time RT-PCR reaction mix revealed no positive signal. Negative (sterile water) and positive controls (synthetic template dilutions) were added systematically to each amplification run.

Among the 671 plasma samples tested, no positive signal was identified using the XMRV gag and pol assays; such negative results were confirmed following agarose gel electrophoresis of real-time amplification products.

Our study involved one of the largest cohorts so far investigated for the prevalence of XMRV and was also designed with a view to optimising the extraction/detection procedure (large plasma volumes, no freezing/thawing cycles). In accordance with studies published in recent months, these results highlight the fact that the virus is not detectable (or extremely rare) in healthy French individuals. Recent publications emphasised the recombinant origin of the virus and that the high prevalences of XMRV found in some early studies were related to laboratory and/or amplification mix contamination, leading to artificially raised prevalence figures. Only studies based on validated protocols will be able to provide incontrovertible information about the natural history of XMRV and its distribution in humans.