Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences

Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
Retrovirology (Impact Factor: 4.19). 12/2010; 7(1):109. DOI: 10.1186/1742-4690-7-109
Source: PubMed


In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections.
DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 μg of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay.
Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.

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    • "IAP are retrotransposon elements present at the level of about 1000 copies of varying length per mouse genome. IAP PCR has been proposed as an assay for the detection of murine DNA contamination of human DNA specimens [33]. Amplification of the IAP sequences from the mouse genome by PCR was carried out using the following primers: forward primer (5′-ATAATCTGCGCATGAGCCAAGG-3′) and reverse primer (5′-AGGAAGAACACCACAGACCAGA-3) under the recommended conditions [33]: one cycle of 95°C, 5 minutes, 35 cycles of 95°C 30 seconds, 58°C 30 seconds, 72°C 20 seconds and one cycle of 72°C, 7 minutes. "
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    ABSTRACT: Background Retroviral sequences 90-95% homologous to the mouse mammary tumor virus (MMTV) were present in 38% of the breast cancers studied from American women and were not detectable in non-tumor breast tissue from the same patient. The entire proviral structure was described and viral particles were isolated from primary cultures of human breast cancer. This virus was designated as human mammary tumor virus (HMTV). Hormone response elements present in the HMTV Long-Terminal-Repeat (LTR) suggest a mechanism for association of HMTV with hormonally responding tissues. In fact, the incidence of HMTV sequences is higher in gestational breast cancers, which are associated with hormonal changes. Milk epithelial cells are also under hormonal regulation and therefore are excellent specimens for HMTV sequence detection. Methods The HMTV sequence was studied in milk samples from lactating women recruited with increased risk of breast cancer because they had undergone breast biopsies (Biopsy-Group) and lactating women without breast biopsies (Reference-Group). Results HMTV-env sequences were detected by PCR in milk of 7.61% of 92 women of the Reference-Group and in 20.55% of 73 women of the Biopsy-Group (p: 0.015). The sequences were 94-98% homologous to MMTV. HMTV-env and HMTV-env/LTR junction sequences were detected in high-speed pellet RNA, implying the presence of HMTV viral particles. PCR assays to detect the murine mitochondrial cytochrome oxidase gene and intracisternal-A-type particle sequences were performed to rule out mouse mitochondrial or genomic DNA contamination. Eight women of the 73 Biopsy-Group participants had breast cancer and the milk of only one of these eight women had HMTV-env sequences. In the remaining 65 women of the Biopsy-Group, under enough clinical suspicion to lead to biopsy, HMTV was detected in 14, nearly three times the number of milks as compared to the Reference-Group (21.54% versus 7.61%; p: 0.016). Conclusion The significance of HMTV in milk from the Reference-Group, the greater frequency in the milk of women who had undergone a breast biopsy and its possible infectivity for infants are important questions under study. The similarity of HMTV to MMTV is striking and suggests one possible avenue for viral transmission in humans.
    Full-text · Article · Jun 2014 · Infectious Agents and Cancer
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    • "Brightness and contrast were lightly adjusted after merging. Image analysis was performed with Image J [10,33] using the JACoP plugin [12,13,34] for quantification of percent cell coverage and manual analysis for vessel density. Confocal images were acquired using a Zeiss LSM700 scanning confocal microscope with 405nm, 488nm, 555nm, and 637nm solid-state lasers. "
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    ABSTRACT: Background Xenotropic Murine leukemia virus-Related Virus (XMRV) is a γ-retrovirus initially reported to be present within familial human prostate tumors and the blood of patients with chronic fatigue syndrome. Subsequent studies however were unable to replicate these findings, and there is now compelling evidence that the virus evolved through rare retroviral recombination events in human tumor cell lines established through murine xenograft experiments. There is also no direct evidence that XMRV infection has any functional effects that contribute to tumor pathogenesis. Results Herein we describe an additional xenotropic MLV, “B4rv”, found in a cell line derived from xenograft experiments with the human prostate cancer LNCaP cell line. When injected subcutaneously in nude mice, LNCaP cells infected with XMRV or B4rv formed larger tumors that were highly hemorrhagic and displayed poor pericyte/smooth muscle cell (SMC) investment, markers of increased metastatic potential. Conditioned media derived from XMRV- or B4rv-infected LNCaPs, but not an amphotropic MLV control virus infected LNCaPs, profoundly decreased expression of marker genes in cultured SMC, consistent with inhibition of SMC differentiation/maturation. Similar effects were seen with a chimeric virus of the amphotropic MLV control virus containing the XMRV env gene, but not with an XMRV chimeric virus containing the amphotropic MLV env gene. UV-inactivated XMRV and pseudovirions that were pseudotyped with XMRV envelope protein also produce conditioned media that down-regulated SMC marker gene expression in vitro. Conclusions Together these results indicate that xenotropic MLV envelope proteins are sufficient to induce the production of factors by tumor cells that suppress vascular SMC differentiation, providing evidence for a novel mechanism by which xenotropic MLVs might alter tumor pathogenesis by disrupting tumor vascular maturation. Although it is highly unlikely that either XMRV or B4Rv themselves infect humans and are pathogenic, the results suggest that xenograft approaches commonly used in the study of human cancer promote the evolution of novel retroviruses with pathogenic properties.
    Full-text · Article · Mar 2013 · Retrovirology
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    • "While the reason for the reported disparity of XMRV prevalence in PC was unclear, it was initially attributed to differences in geography or assay sensitivity. However, subsequent studies demonstrated that positive signals in sensitive PCR assays could be ascribed to either mouse DNA contamination or contamination with XMRV DNA from a commonly used PC cell line (22Rv1), which harbours 10–20 copies of XMRV [20-26]. The discovery that XMRV was generated by recombination of two mouse endogenous retroviruses following passage of a PC xenograft in nude mouse demonstrated that XMRV was generated in the laboratory [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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