XMRV Infection Research Articles

Correction: Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

The authors wish to retract this article for the following reasons: On re-examination of the data, some errors were identified which contradicted some of the main conclusions of the work. In particular, an error in the calculation of ∆(-) values for miR-193a-3p and miRPlus E1245 was identified, which affects the results reported in Figure 6, and hence the data does not support the conclusions originally drawn regarding the E1245. In addition, the interpretation of the presented PCA results is inaccurate since there is no convincing evidence of large or consistent variation related to treatment effect. Since the experiments were performed only twice, there is no statistical support for the reported miRNA expression level changes during XMRV infection of human cell, and therefore the conclusion that XMRV infection modulates miRNAs in host cells cannot be reported with confidence unless supported by additional experimental validation.

Xenotropic MLV envelope proteins induce tumor cells to secrete factors that promote the formation of immature blood vessels

BackgroundXenotropic 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.ResultsHerein 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.ConclusionsTogether 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.

Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection

Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.

In-depth investigation of archival and prospectively collected samples reveals no evidence for XMRV infection in prostate cancer.

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

Identification of XMRV infection-associated microRNAs in four cell types in culture.

IntroductionXMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.MethodsTwo prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.ResultsMicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.DiscussionThe present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.

Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).

Xpr1 Is an Atypical G-Protein-Coupled Receptor That Mediates Xenotropic and Polytropic Murine Retrovirus Neurotoxicity

Xenotropic murine leukemia virus-related virus (XMRV) was first identified in human prostate cancer tissue and was later found in a high percentage of humans with chronic fatigue syndrome (CFS). While exploring potential disease mechanisms, we found that XMRV infection induced apoptosis in SY5Y human neuroblastoma cells, suggesting a mechanism for the neuromuscular pathology seen in CFS. Several lines of evidence show that the cell entry receptor for XMRV, Xpr1, mediates this effect, and chemical cross-linking studies show that Xpr1 is associated with the Gβ subunit of the G-protein heterotrimer. The activation of adenylate cyclase rescued the cells from XMRV toxicity, indicating that toxicity resulted from reduced G-protein-mediated cyclic AMP (cAMP) signaling. Some proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found no role of Xpr1 in phosphate uptake or its regulation. Our results indicate that Xpr1 is a novel, atypical G-protein-coupled receptor (GPCR) and that xenotropic or polytropic retrovirus binding can disrupt the cAMP-mediated signaling function of Xpr1, leading to the apoptosis of infected cells. We show that this pathway is also responsible for the classic toxicity of the polytropic mink cell focus-forming (MCF) retrovirus in mink cells. Although it now seems clear that the detection of XMRV in humans was the result of sample contamination with a recombinant mouse virus, our findings may have relevance to neurologic disease induced by MCF retroviruses in mice.

Absence of Detectable XMRV and Other MLV-Related Viruses in Healthy Blood Donors in the United States

BackgroundPreliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.Methodology/Principal FindingsTo gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.Conclusions/SignificanceOur results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.

Characterization, Mapping, and Distribution of the Two XMRV Parental Proviruses

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.

XMRV: usage of receptors and potential co-receptors

BackgroundXMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors.MethodsTo study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR.ResultsInfection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP.ConclusionXMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.

Heme oxygenase-1 activation inhibits XMRV pathogenesis and carcinogenesis in prostate cancer cells

Heme oxygenase-1 (HO-1) induction by hemin, the active ingredient of an FDA-approved biologic for the treatment of acute porphyries, has been previously shown in our laboratory to effectively inhibit HIV-1 replication. The present study was undertaken to expand these observations to examine the inhibitory role of HO-1 in the pathogenesis of XMRV infection. Hemin-induced HO-1 activation in LNCaP, a prostate cancer cell line susceptible to XMRV infection, markedly down-modulated the cell surface expression of XMRV receptor Xpr1, and significantly inhibited their ability to support productive virus replication. Hemin treatment of XMRV-integrated prostate carcinoma cells 22Rv1 was relatively less effective in suppressing the rapidly replicating XMRV in these aggressive malignant cells; yet it efficiently inhibited XMRV infection of LNCaP cells by about 80% when cultured for three days in the virus-containing 50% conditioned media from 22Rv1 cells. Additionally, HO-1 induction retarded the growth of uninfected LNCaP cells, XMRV-infected LNCaP cells and 22Rv1 cells, and significantly reduced their invasiveness to the reconstituted basement membrane matrix Matrigel, consistent with the lower levels of basement membrane degrading enzyme matrix metalloproteinase-9. These findings indicate a pivotal role of HO-1 as a host cell defense mechanism in prostate carcinogenesis in vitro, and may offer HO-1 induction as a potentially novel therapeutic strategy to control the pathogenesis of XMRV infection.

Immune correlates of XMRV infection

Methods This hypothesis was addressed by multiplex profiling of plasma cytokines and chemokines on a LuminexTM platform and phenotypic analysis of leukocyte subsets by multi-parameter flow cytometry in XMRV infected CFS patients versus uninfected controls. XMRV-infected subject and control samples were assayed blindly. Analysis was performed using the Gene Expression Pattern Analysis Suite and Random Forest tree classification algorithms. For immune profiling, 63 XMRV infected CFS patient samples were analyzed within 6 hours using an LSRII flow cytometer with BD FACSDiva software. Six normal donors and reference values based on a healthy population were used as normal baselines. Results

Development of XMRV producing B Cell lines from lymphomas from patients with Chronic Fatigue Syndrome

Previous studies have shown that CFS patients have an increased incidence of lympho-proliferative malignancy compared to the normal population [1]. While the incidence rate of non-Hodgkin’s lymphoma in the United States is 0.02%, nearly 5% of the CFS patients developed the disease. Additionally, development of cancer coincides with an outgrowth of gamma delta T cells with specific clonal T-cell receptor gamma rearrangements. We hypothesized that infection with XMRV and/or other viruses can trigger a dysregulated immune response which favors the development of B-cell lymphoma. In a study of 300 CFS patients, 13 developed lymphoproliferative disorders. Of those tested, 11/11 were positive for XMRV and 9/9 positive for clonal TCR gamma rearrangements. Spontaneous development of four B cells lines occurred during culture of cells from CFS patients. Three developed from B cells isolated from the peripheral blood (two of whom had B cell lymphoma) and one from a bone marrow biopsy. For all four lines, the B cells have a mature CD20+, CD23+ phenotype and produce infectious XMRV at a titer of >106 infectious units/ml. Virus production occurred despite extensive hypermutation of the proviruses in these cells by APOBEC3G.. Therefore XMRV infection may accelerate the development of B cell malignancies by either indirect chronic stimulation of the immune system and/or by direct infection of the B-cell lineage . Since viral load in peripheral blood is low, these data suggest that B cells in tissues such as spleen and lymph nodes could be an in vivo reservoir for XMRV.

Abstract LB-433: Examination of the oncogenic potential of XMRV in prostate epithelial cells

Abstract Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus closely related to murine leukemia viruses, which cause a variety of cancers and immunological and neurological diseases in mice. Whether XMRV plays a role in cancer and other diseases remains controversial; some studies show a possible association, but there is currently no evidence that the virus is a causal agent for disease. Our hypothesis was that the 3′ long terminal repeat (LTR) from the XMRV genome is responsible for its oncogenic activity in prostate cells. We chose three regions from the XMRV 3′ LTR and tested the potential of these regions in transactivating a cellular gene promoter whose homolog is transactivated by MMLV in mouse cells. We examined the transactivation potential in pre-cancerous and a panel of cancerous prostate epithelial cells. Our results showed that for certain 3′LTR regions induce nearly full transactivating activity, while others might comprise inhibitory regions. Therefore, to better understand the mechanism by which the XMRV 3′ LTR regions might be inducing or inhibiting transactivation, we tested their ability to transactivate cellular genes related to transformation. We used as reporters plasmids containing binding sites for cellular transcription factors. The results showed several similarities in transactivation, suggesting common activation pathways for 3′LTR transactivating activity. Finally, we tested if XMRV infection could enhance oncogenicity potential of prostate precancerous and cancerous cell lines. We examined this in vitro by assessing the rate of colony formation in soft agar, and in vivo, using xenograft assays. The results suggested that cell lines infected with XMRV had enhanced in vitro colony formation, as well as enhanced in vivo growth, however, addition of another tumorigenic genetic alteration was required in conjunction with XMRV-infection to induce enhanced tumor growth. Our data suggests that the XMRV 3′LTR may have a transactivating activity on cellular genes, and that XMRV might have a transforming activity in prostate cancer cells, most likely stimulating promotion/progression of tumorigenesis, and not ‘initiation’ of tumorigenesis. These results are highly novel since they represent for the first examinations of the role of XMRV in tumorigenesis of prostate epithelial cell lines in vitro and in vivo and examinations of the 3′LTR context of the virus upon transactivation of cellular genes as a potential mechanism of cellular transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-433. doi:10.1158/1538-7445.AM2011-LB-433

No evidence of XMRV in prostate cancer cohorts in the Midwestern United States

BackgroundXenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies.ResultsReal-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution.ConclusionThe lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.

Infection, Viral Dissemination, and Antibody Responses of Rhesus Macaques Exposed to the Human Gammaretrovirus XMRV

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

Xenotropic and Other Murine Leukemia Virus-Related Viruses in Humans

The recent discovery of a xenotropic murine leukemia virus-related retrovirus (designated as XMRV) in prostate cancer tissues and later in chronic fatigue syndrome (CFS) patients created excitement related to possible association of a virus with these human diseases. However, the failure to reproduce such results in other laboratories raised concerns and much debate among scientists, patient populations, and clinicians over the original findings. This special issue is a collection of recent research reports, that address this controversy. It also includes several expert reviews on various aspects of murine retroviruses, such as virus biology, replication, phylogeny, and pathogenesis, as well as XMRV in prostate cancer. The first paper by A. Rein reviews the murine retrovirus genomic structure, viral structural proteins, and virus replication. The second paper by C. A. Kozak provides a detailed review of murine retrovirus entry into the cell and different receptor usage by different types of murine retroviruses with comparison to XMRV. The third paper by J. Blomberg et al. discusses the phylogenetic analysis of murine retroviruses and other retroviruses. The fourth paper by J. Chakraborty et al. discusses murine retrovirus pathogenesis and a mouse model for transmission of lymphoma by breast milk. The fifth paper by D. E. Kang et al. reviews the discovery, progress, and current status of XMRV findings in prostate cancer patients. The sixth paper by S. Tang and I. K. Hewlett reviews XMRV detection assays and deficiencies in the testing methods. The seventh paper by J. M. Coffin and O. Cingoz provides a detailed review of the controversies related to the XMRV results in human clinical samples and the findings regarding virus origin and discusses the potential sources of contamination that resulted in the misidentification of the virus as a novel human retrovirus. Additionally, the special issue contains various research papers demonstrating the absence of XMRV in various patient populations using sensitive assays for virus detection. The eighth paper by B. Oakes et al. reports the absence of antibodies in CFS patients and healthy controls using two novel sensitive immunoassays. The ninth paper by J. Spindler et al. reports the lack of evidence of XMRV infection in HIV-1 infected men or men at high risk for HIV-1 infection by analyzing PBMCs and plasma samples using sensitive PCR assays and immunoassays. The tenth paper by K. A. Delviks-Frankenberry et al. demonstrates the absence of XMRV in PBMCs and plasma from HIV-1 lymphoma patients using PCR or immunoassays. The eleventh paper by M. J. Robinson et al. indicates the absence of XMRV sequences in prostate cancer samples from diverse populations, B-cell lymphoma patients, as well as UK blood donors. The twelfth paper by M. Kearney et al. reports the use of different methodologies to demonstrate the absence of XMRV in plasma and in some tissue samples from prostate cancer patients. The final, thirteenth paper by P. Sharma et al. describes XMRV infection in the reproductive tissues of rhesus monkeys, proposing the possibility of an animal model for further investigations of virus transmission. This special issue provides the recent thinking and research results of studies on XMRV and other murine leukemia retrovirus-related sequences in humans. Numerous studies have failed to confirm the presence of XMRV in humans, and XMRV has recently been found to be a laboratory-derived rare recombinant, which originated by xenografting a patient's prostate cancer cells in nude mice. A consensus has now emerged that XMRV footprints or infectious XMRV detected in normal human individuals or in some diseased patients represents laboratory contaminations. The information provided in this issue should be of interest to a broad audience including scientists, clinicians, patient populations, and public health agencies. Arifa S. Khan Myra McClure Yoshinao Kubo Paul Jolicoeur

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

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.

Apobec 3G Efficiently Reduces Infectivity of the Human Exogenous Gammaretrovirus XMRV

BackgroundThe human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.Methodology/Principal FindingsHere we demonstrate that XMRV is resistant to human Apobec 3B, 3C and 3F, while being highly susceptible to the human A3G protein, a factor which is known to confer antiviral activity against most retroviruses. We show that XMRV as well as MoMLV virions package Apobec proteins independent of their specific restriction activity. hA3G was found to be a potent inhibitor of XMRV as well as of MoMLV infectivity. In contrast to MoMLV, XMRV infection can also be partially reduced by low concentrations of mA3. Interestingly, established prostate cancer cell lines, which are highly susceptible to XMRV infection, do not or only weakly express hA3G.ConclusionsOur findings confirm and extend recently published data that show restriction of XMRV infection by hA3G. The results will be of value to explore which cells are infected with XMRV and efficiently support viral spread in vivo. Furthermore, the observation that XMRV infection can be reduced by mA3 is of interest with regard to the current natural reservoir of XMRV infection.

Absence of evidence of Xenotropic Murine Leukemia Virus-related virus infection in persons with Chronic Fatigue Syndrome and healthy controls in the United States

BackgroundXMRV, a xenotropic murine leukemia virus (MuLV)-related virus, was recently identified by PCR testing in 67% of persons with chronic fatigue syndrome (CFS) and in 3.7% of healthy persons from the United States. To investigate the association of XMRV with CFS we tested blood specimens from 51 persons with CFS and 56 healthy persons from the US for evidence of XMRV infection by using serologic and molecular assays. Blinded PCR and serologic testing were performed at the US Centers for Disease Control and Prevention (CDC) and at two additional laboratories.ResultsArchived blood specimens were tested from persons with CFS defined by the 1994 international research case definition and matched healthy controls from Wichita, Kansas and metropolitan, urban, and rural Georgia populations. Serologic testing at CDC utilized a Western blot (WB) assay that showed excellent sensitivity to MuLV and XMRV polyclonal or monoclonal antibodies, and no reactivity on sera from 121 US blood donors or 26 HTLV-and HIV-infected sera. Plasma from 51 CFS cases and plasma from 53 controls were all WB negative. Additional blinded screening of the 51 cases and 53 controls at the Robert Koch Institute using an ELISA employing recombinant Gag and Env XMRV proteins identified weak seroreactivity in one CFS case and a healthy control, which was not confirmed by immunofluorescence. PCR testing at CDC employed a gag and a pol nested PCR assay with a detection threshold of 10 copies in 1 ug of human DNA. DNA specimens from 50 CFS patients and 56 controls and 41 US blood donors were all PCR-negative. Blinded testing by a second nested gag PCR assay at the Blood Systems Research Institute was also negative for DNA specimens from the 50 CFS cases and 56 controls.ConclusionsWe did not find any evidence of infection with XMRV in our U.S. study population of CFS patients or healthy controls by using multiple molecular and serologic assays. These data do not support an association of XMRV with CFS.