Analysis of XMRV integration sites from prostate cancer tissues suggests PCR contamination rather than genuine human infection

MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland St, London W1T 4JF, UK.
Retrovirology (Impact Factor: 4.19). 02/2011; 8(article 13):13. DOI: 10.1186/1742-4690-8-13
Source: PubMed


XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells. Identical integration sites have never previously been described in any retrovirus infection. We propose that the patient-derived sites are the result of PCR contamination. This observation further undermines the notion that XMRV is a genuine human pathogen.

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Available from: Paul Kellam, Dec 20, 2014
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    • "Phylogenetic analysis of XMRV sequences from unlinked patients and a commonly used PC cell line (22Rv1) showed that these sequences formed a monophyletic clade and that the cell line-derived sequences were ancestral to the patient-derived sequences (posterior probability >0.99). These findings led to the conclusion that XMRV contamination originated from the 22Rv1 cell line [25,26]. Furthermore, the possibility that XMRV in 22Rv1 cells originated from a bone fide human infection was debunked by Paprotka and colleagues who showed that XMRV was a laboratory virus generated by a rare recombination event between two mouse endogenous retroviruses during passage of the CWR22 PC xenograft in nude mice from which the 22Rv1 cell line was derived [27]. "
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    ABSTRACT: Background Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). While the association of XMRV with CFS and PC has recently been discredited, no studies have been performed in Australian patients to investigate the association between PC and XMRV or related murine leukemia virus (MLV) in matched PC and normal tissue. Methods Genomic DNA (gDNA) was purified from matched normal and cancer formalin-fixed paraffin-embedded (FFPE) prostate tissue from 35 Australian PC patients with Gleason scores ranging from 7 – 10. The presence of the ribonuclease L (RNase L) polymorphism R462Q was determined by allele specific PCR. Samples were screened for XMRV and related murine leukemia virus (MLV) variants by qPCR. Contaminating mouse DNA was detected using qPCR targeting mouse intracisternal A particle long terminal repeat DNA. Results gDNA was successfully purified from 94% (66/70) of normal and cancer FFPE prostate tissues. RNase L typing revealed 8% were homozygous (QQ), 60% were heterozygous (RQ) and 32% were wild-type (RR) for the RNase L mutation. None of the 66 samples tested were positive for XMRV or related MLV sequences using broad MLV or XMRV specific primers with detection sensitivities of 1 viral copy of MLV/XMRV and XMRV DNA, respectively. Conclusions Using highly sensitive qPCR we found no evidence of XMRV or related gammaretroviruses in prostate tissues from 35 Australian PC patients. Our findings are consistent with other studies demonstrating that XMRV is a laboratory contaminant that has no role in the aetiology of PC.
    Full-text · Article · Jan 2013 · Virology Journal
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    • "XMRV has been originally identified in prostate tissue from patients with familial prostate cancer [8]; subsequent work provided evidence of XMRV protein expression in up to 23% of all prostate cancer cases [9]. However, multiple studies failed to detect XMRV in prostate cancer samples using PCR or IHC methods [10], [11], [12], [13], [14], [15], [16], [17], [18], [19] Due to the lack of sequence variability of XMRV gene fragments in patients’ isolates compared to sequence variability identified in a XMRV positive cell line 22Rv1 it was postulated that XMRV might be a laboratory contaminant rather than a true exogenous human virus [20]. These data are strengthened by recent data of Paprotka and colleagues analysing different passages of CWR22 xenografts: XMRV is present in 22Rv1 cells and CWR-R1 cells, however, early passages of the CWR xenograft do not carry any detectable XMRV sequences. "
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    ABSTRACT: 22Rv1 is a common prostate cancer cell line used in xenograft mouse experiments as well as in vitro cell culture assays to study aspects of prostate cancer tumorigenesis. Recently, this cell line was shown to harbor multiple copies of a gammaretrovirus, called XMRV, integrated in its genome. While the original prostate cancer xenograft CWR22 is free of any retrovirus, subsequently generated cell lines 22Rv1 and CWR-R1, carry this virus and additionally shed infectious gammaretroviral particles in their supernatant. Although XMRV most likely was generated by recombination events in cell culture this virus has been demonstrated to infect human cells in vitro and 22Rv1 as well as CWR-R1 cells are now considered biosafety 2 reagents. Here, we demonstrate that 22Rv1 cells with reduced retroviral transcription show reduced tumor angiogenesis and increased necrosis of the primary tumor derived from xenografted cells in scid mice when compared to the parental cell line. The presence of XMRV transcripts significantly increases secretion of osteopontin (OPN), CXCL14, IL13 and TIMP2 in 22Rv1 cells. Furthermore, these data are supported by in vitro cell invasion and differentiation assays. Collectively, our data suggest that the presence of XMRV transcripts at least partially contributes to 22Rv1 characteristics observed in vitro and in vivo with regard to migration, invasion and tumor angiogenesis. We propose that data received with 22Rv1 cells or equivalent cells carrying xenotropic gammaretroviruses should be carefully controlled including other prostate cancer cell lines tested for viral sequences.
    Full-text · Article · Jul 2012 · PLoS ONE
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    • "In order to explain the positive results, several studies suggested that the detection of XMRV sequences in human samples was a result of contamination of laboratory reagents with mouse DNA [36]–[44], with DNA from the chronically infected DU145 cell line [45], [46] or with a XMRV plasmid DNA [33]. Finally, the assertion that XMRV is circulating in human population has been challenged by the report showing that XMRV was generated by a recombination event between two endogenous MLVs during in vivo tumor passaging in mice [47], which yielded the popular prostate cancer cell line 22Rv1 [48]. "
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    ABSTRACT: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus.
    Full-text · Article · May 2012 · PLoS ONE
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