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An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

Laboratory of Signal Transduction, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan.
Retrovirology (Impact Factor: 4.77). 12/2010; 7(1, article 110):110. DOI: 10.1186/1742-4690-7-110
Source: PubMed

ABSTRACT During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.

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    • "However, gammaretroviruses are known to induce cancer in animals, understanding XMRV or related MLV infections in human prostate cancer tissues will shed light on their potential contribution to human disease. Proposed reasons to explain the conflicting data are unknown but may be: technical differences, lack of standardized XMRV PCR assays, assay sensitivity, contamination by and cross-reactivity of XMRV PCR assays with closely related endogenous MLVs such as trace quantities of mouse genomic DNA found in reagents and samples (Hue et al., 2010; Oakes et al., 2010; Robinson et al., 2010; Sato et al., 2010; Knox, Carrigan et al., 2011; Tuke et al., 2011), differences in the geographical distribution of XMRV, sequence differences among XMRV genomes (Silverman et al., 2010; Singh et al., 2010; Knox et al., 2011) and factors related to the population genetic factors (Switzer et al., 2011). Due to public health and medical consequences of potential XMRV or related MLVs infection in humans, we considered it is important to confirm or reject their association with prostate cancer in Iranian context. "
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    ABSTRACT: Background: Multiple etiologies have been hypothesized for prostate cancer, including genetic defects and infectious agents. A recently reported gamaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) has been reported to be detected in prostate cancer. However, this virus has not been detected in similar groups of patients in other studies. Herein, we sought to detect XMRV in prostate cancers and benign controls in Sanandaj, west of Iran. Materials and Methods: In a case-control study, genomic DNA was extracted from formalin fixed and paraffin embedded prostate tissues from a total of 163 Iranian patients. We developed a conventional and a nested PCR assay using primers targeting to an env specific sequence of XMRV. PCR assays were carried out on 63 prostate cancers and 100 benign prostate hyperplasias. Results: Beta-actin sequences were successfully detected in the DNA extracts from all prostate tissues, confirming DNA extraction integrity. We did not detect XMRV in samples either from prostate cancers or benign prostate hyperplasias using XMRV specific primers. Conclusions: We conclude that in our population XMRV does not play a role in genesis of prostate cancer.
    Asian Pacific journal of cancer prevention: APJCP 11/2013; 14(11):6929-33. DOI:10.7314/APJCP.2013.14.11.6929 · 2.51 Impact Factor
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    • "Similarly, a role for XMRV in the pathogenesis of chronic fatigue syndrome was also reported (Lombardi et al, 2009), but this association has now been discredited and retracted (van der Meer et al, 2010; Paprotka et al, 2011; Steffen et al, 2011; Alberts, 2011). Furthermore, some studies have reported that XMRV is not an exogenous virus at all, but rather a mouse endogenous virus contaminant (Hue et al, 2010; Sato et al, 2010; Smith, 2010). "
    British Journal of Cancer 03/2012; · 4.82 Impact Factor
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    • "Similarly, a role for XMRV in the pathogenesis of chronic fatigue syndrome was also reported (Lombardi et al, 2009), but this association has now been discredited and retracted (van der Meer et al, 2010; Paprotka et al, 2011; Steffen et al, 2011; Alberts, 2011). Furthermore, some studies have reported that XMRV is not an exogenous virus at all, but rather a mouse endogenous virus contaminant (Hue et al, 2010; Sato et al, 2010; Smith, 2010). "
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    ABSTRACT: The aetiology of breast cancer remains elusive. A viral aetiology has been proposed, but to date no virus has been conclusively demonstrated to be involved. Recently, two new viruses, namely Merkel cell polyomavirus (MCV) and xenotropic murine leukaemia virus-related virus (XMRV) have been identified and implicated in the pathogenesis of Merkel cell carcinoma (MCC) and familial form of prostate cancer, respectively. We examined 204 samples from 58 different cases of breast cancer for presence of MCV or XMRV by PCR. Samples consisted of both malignant and non-malignant tissues. Additionally, we included 6 cases of MCC and 12 cases of prostate cancer as potential controls for MCV and XMRV, respectively. All of the breast cancer samples examined were negative for both MCV and XMRV. However, 4/6 MCC and 2/12 prostate cancer samples were found to be positive for MCV and XMRV, respectively. Sequence analysis of the amplified products confirmed that these sequences belonged to MCV and XMRV. We conclude that there is no evidence for the involvement of MCV or XMRV in the pathogenesis of breast cancer. What role these viruses have in the pathogenesis of MCC and prostate carcinomas remains to be demonstrated.
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