Xenotropic Murine Leukemia Virus-related Virus Sequences Research Articles

No association found between the detection of either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus and chronic fatigue syndrome in a blinded, multi-site, prospective study by the establishment and use of the SolveCFS BioBank.

BackgroundIn 2009, a retrospective study reported the detection of xenotropic murine leukemia virus-related virus (XMRV) in clinical isolates derived from individuals with chronic fatigue syndrome or myalgic encephalomyelitis (CFS). While many efforts to confirm this observation failed, one report detected polytropic murine leukemia virus (pMLV), instead of XMRV. In both studies, Polymerase Chain Reaction (PCR)-based methods were employed which could provide the basis for the development of a practical diagnostic tool. To confirm these studies, we hypothesized that the ability to detect these viruses will not only depend upon the technical details of the methods employed but also on the criteria used to diagnose CFS and the availability of well characterized clinical isolates.MethodsA repository of clinical isolates from geographically distinct sites was generated by the collection of fresh blood samples from well characterized CFS and healthy subjects. Molecular techniques were used to generate assay positive controls and to determine the lower limit of detection (LLOD) for murine retroviral and Intracisternal A particle (Cell 12(4):963-72, 1977) detection methods.ResultsWe report the establishment of a repository of well-defined, clinical isolates from five, geographically distinct regions of the US, the comparative determination of the LLODs and validation efforts for the previously reported detection methods and the results of an effort to confirm the association of these retroviral signatures in isolates from individuals with CFS in a blinded, multi-site, prospective study. We detected various, murine retroviral DNA signatures but were unable to resolve a difference in the incidence of their detection between isolates from CFS (5/72; 6.7%) and healthy (2/37; 5.4%) subjects (Fisher’s Exact Test, p-value = 1). The observed sequences appeared to reflect the detection of endogenous murine retroviral DNA, which was not identical to either XMRV or pMLV.ConclusionsWe were unable to confirm a previously reported association between the detection of XMRV or pMLV sequences and CFS in a prospective, multi-site study. Murine retroviral sequences were detected at a low frequency that did not differ between CFS and control subjects. The nature of these sequences appeared to reflect the detection of pre-existing, endogenous, murine retroviral DNA in the PCR reagents employed.

Lack of the Detection of XMRV or Polytropic MLV-Related Sequences in Blood Cells From HIV-1–Infected Patients in Spain

Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus. Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination. No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed. XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.

The scientific method at work: xenotropic murine leukemia virus–related virus is neither a cause of chronic fatigue syndrome nor a threat to the blood supply

S ir Francis Bacon (1561-1626), the Lord Chancellor of England, was the first to provide a documented philosophical method for investigating a natural phenomenon. This method later became known as “the scientific method.” Unlike many before him, he suggested that our understanding of the world should be based on data, rather than faith or dogma. Moreover, his method required that our understanding of the world be provisional, with the hope that our current understanding of natural phenomena would eventually be replaced by better science. The events that have transpired over the past 2 years regarding the clinical relevance of xenotropic murine leukemia virus–related virus (XMRV) have shown that the method of scientific investigation first described 400 years ago is very much alive and well today. The scientific method, as envisioned by Sir Francis Bacon, starts with observation. Urisman and colleagues first observed that XMRV is associated with human disease in 2006, finding that 40% of men with prostate cancer and a low activity variant of RNase L, an enzyme involved in the interferon-induced antiviral response, were infected with this virus. Subsequently in 2009 and 2010, two groups of investigators also described finding XMRV or related sequences of polytropic murine leukemia viruses (MLVs) in association with chronic fatigue syndrome (CFS), a disease characterized by severe fatigue and other related symptoms lasting more than 6 months. XMRV is currently understood to be a retrovirus, but is unrelated to other well-described retroviruses such as human immunodeficiency virus (HIV) and human T-cell lymphotropic virus (HTLV). XMRV specifically is a member of the family Retroviridae, subfamily Orthoretrovirinae, and genus Gammaretrovirus (see AABB XMRV Fact Sheet http://www.aabb.org/resources/bct/ eid/Pages/default.aspx). XMRV is the first gammaretrovirus to be found in humans, and data indicate that it originated in mice after the recombination of two murine proviruses. Virions are 80 to 100 nm in diameter, consisting of an envelope, nucleocapsid, and a nucleoid with a linear dimer of positive-sense, single-stranded RNA. From these initial observations, it was hypothesized that this virus could be causally related to both prostate cancer and CFS. Moreover, the finding of viral sequences in the blood of healthy controls in two studies led to the concern that this virus could be transfusion transmitted, and thus the national blood supply could be at risk. Specifically, on June 18, 2010, the AABB issued a bulletin to its membership from its Interorganizational Task Force on XMRV that patients diagnosed with CFS be discouraged from donating blood. Consequently, the American Red Cross and a number of other blood donor centers started to offer educational information about CFS and have requested voluntary deferral of donors who ever have had a medical diagnosis of this debilitating condition. While the risk of transmission of XMRV by blood products was unknown at the time of release of this bulletin, the recommendation of the AABB Interorganizational Task Force was reasonable based on the initial hypothesis that CFS could have an infectious origin. First, XMRV is a gammaretrovirus, a genus that contains known animal pathogens (see AABB XMRV Fact Sheet). As other retroviruses, such as HIV and HTLV, are transfusion transmitted, it was plausible that an emerging retrovirus, such as XMRV, could also be transmitted by blood. Second, studies indicated that XMRV was physically present in blood. A rhesus macaque model of XMRV previously demonstrated that the virus can infect lymphoid cells, several tissues, and organs even though circulation of free virus was minimal. Moreover, Lombardi and colleagues found XMRV infection in the lymphocytes of the CFS patients From the Department of Pathology, Johns Hopkins University, Baltimore, Maryland; and the Scientific Support Office, American Red Cross, Gaithersburg, Maryland. Address reprint requests to: Susan Stramer, Scientific Support Office, American Red Cross, 9315 Gaither Road, Gaithersburg, MD 20877; e-mail: stramers@usa.redcross.org. Received for publication November 28, 2011; accepted November 28, 2011. doi: 10.1111/j.1537-2995.2011.03518.x TRANSFUSION 2012;52:222-225.

No evidence of cross‐species transmission of mouse retroviruses to animal workers exposed to mice

Although recent data have brought into question the association between xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome, one group has reported evidence of human infection with distinct polytropic murine leukemia viruses (MLVs). Occult retroviral infection among humans poses a significant public health risk should it be introduced into the blood supply. To explore the possibility of cross-species transmission of MLVs to humans, we sought molecular and serologic evidence of XRMV/MLV infection among a cohort of animal workers highly exposed to mice. Before the commencement of the study, the laboratory and equipment were demonstrated to be free of XMRV/MLV DNA sequences. DNA extracted from 43 animal workers was tested using nested polymerase chain reaction (PCR) with published primer sets, targeting regions of XMRV and MLV gag. Negative controls were assayed in a 1:1 ratio with specimens. Serum specimens were tested using a validated immunoblot assay containing cross-reactive XMRV antigens. Initial molecular assays demonstrated that the physical space and laboratory equipment were free of MLV and XMRV DNA sequences. Nested PCR assays using multiple primer sets successfully amplified XMRV and MLV sequences from positive controls with high sensitivity. A single, nonreproducible, false-positive result from one specimen was shown to be the result of subsequent contamination. Immunoblotting of all subjects' sera failed to demonstrate any evidence of seroreactivity to XMRV proteins. There was no evidence of human infection with XMRV/MLV among a cohort of individuals highly exposed to mice. These data suggest that the likelihood of cross-species transmission events of MLV from mice to humans is low.

Seroprevalence of xenotropic murine leukemia virus–related virus in normal and retrovirus‐infected blood donors

Xenotropic murine leukemia virus-related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection. Plasma from 1000 US, 100 human immunodeficiency virus Type 1-infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction. XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I-infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I-infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal. Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies.

Xenotropic murine leukemia virus-related virus is not associated with chronic fatigue syndrome in patients from different areas of the us in the 1990s.

BackgroundIn 2009, xenotropic murine leukemia virus-related virus (XMRV) was reported in 67% of patients with chronic fatigue syndrome (CFS) compared to 4% of controls. Since then numerous reports failed to detect XMRV in other cohorts of CFS patients, and some studies suggested that XMRV sequences in human samples might be due to contamination of these samples with mouse DNA.ResultsWe determined the prevalence of XMRV in patients with CFS from similar areas in the United States as the original 2009 study, along with patients with chronic inflammatory disorders and healthy persons. Using quantitative PCR, we initially detected very low level signals for XMRV DNA in 15% of patients with CFS; however, the frequency of PCR positivity was no different between patients with CFS and controls. Repeated attempts to isolate PCR products from these reactions were unsuccessful. These findings were supported by our observations that PHA and IL-2 stimulation of peripheral blood mononuclear cells from patients with apparently low levels of XMRV, which induced virus replication in the 2009 report, resulted in the disappearance of the signal for XMRV DNA in the cells. Immunoprecipitation of XMRV-infected cell lysates using serum from patients from whom we initially detected low levels of XMRV DNA followed by immunoblotting with antibodies to XMRV gp70 protein failed to detect antibody in the patients, although one control had a weak level of reactivity. Diverse murine leukemia virus (MLV) sequences were obtained by nested PCR with a similar frequency in CFS patients and controls. Finally, we did not detect XMRV sequences in patients with several chronic inflammatory disorders including rheumatoid arthritis, Bechet's disease, and systemic lupus erythematosus.ConclusionsWe found no definitive evidence for XMRV DNA sequences or antibody in our cohort of CFS patients, which like the original 2009 study, included patients from diverse regions of the United States. In addition, XMRV was not detected in a cohort of patients with chronic inflammatory disorders.

DNA Extraction Columns Contaminated with Murine Sequences

Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.

Origin of XMRV and its Demise as a Human Pathogen Associated with Chronic Fatigue Syndrome

Retroviruses are well known pathogens of mammals, birds and fish. Their potential to induce cancer in chickens was already described almost 100 years ago and murine retroviruses have been a subject of study for 50 years. The first human retroviruses, HTLV and HIV, were discovered more than 30 years ago, surprising researchers and physicians by the profound differences in the diseases they cause. HTLV-1 is able to induce, after decades of infection, lymphomas/leukemia or neuroimmune disorders whereas untreated HIV infection leads almost inevitably to AIDS. The recently described XMRV (xenotropic murine leukemia virus-related virus) appeared to possess many of the features known for HTLV and was regarded by some to be the third human retrovirus. However, recent publications by Knox et al. [1] and Paprotka et al. [2] have shed new light on this gammaretrovirus. Knox and colleagues clearly demonstrate that XMRV is absent in patients belonging to a chronic fatigue syndrome cohort who had previously been reported to be XMRV-positive [3]. This supports the growing suspicion that laboratory contamination was responsible for the postulated link between XMRV and the disease. Furthermore, Paprotka et al’s identification of XMRV’s origin and the phylogenetic analysis of known XMRV sequences are further nails in the coffin to the notion that XMRV is a clinically relevant infectious human retrovirus.

XMRV: Not a Mousy Virus

The newly discovered mouse-derived human retrovirus, xenotropic murine leukemia virusrelated virus (XMRV), is currently fuelling both the scientific and public debate, where skeptics suggest that results are based on laboratory contamination, while the “believers” have already dressed the virus with highly pathogenic potential. Could the truth lie somewhere in the middle? XMRV was discovered in 2006 in tumor tissue from patients with prostate cancer,1 with a viral genome sequence highly similar to that of mouse xenotropic retroviruses. Sequence analysis suggested that XMRV is a novel recombinant derived from two fragmented endogenous murine viruses integrated in the mouse genome. XMRV was subsequently detected in other prostate cancer tissues and in blood from patients with CFS (chronic fatigue syndrome). However, most other studies failed to replicate these findings, especially outside the USA, suggesting either that the virus has a limited geographical spread, or that positive results were due to contamination of biological reagents or human samples with mouse DNA. Four recent papers indeed show that murine DNA sequences can be detected virtually everywhere,2−5 and that extreme care should be taken when amplifying XMRV sequences. These results certainly put into serious doubt some of the high prevalence results and proposed disease associations that could not be confirmed by others. However, these recent studies do not imply, and in fact did not intend to prove, that all positive results reported thus far are due to contamination, and that XMRV in humans can be dismissed as an artefact. The most important remaining question is: does XMRV infect and replicate in humans, in other words is it a genuine human virus apart from being an easy and frequent contaminant? The relevant subsequent questions would be: “how did XMRV end up in humans” and “is XMRV infection associated with disease”? It is of utmost importance to resolve these questions in a timely manner, as some CFS patients have begun taking antiretroviral drugs that can have side effects. Based on the original XMRV studies, it has also been proposed that CFS patients be banned from donating blood. The best evidence for XMRV replicating in human cells is the detection of proviral integrations flanked by human genome sequences in prostate tissue from eleven patients.6,7 The flanks are human and not mouse sequences, and the insertion sites differ for each clinical sample, thus ruling out a mouse-contamination and

No Xenotropic Murine Leukemia Virus–related Virus Detected in Fibromyalgia Patients

No Xenotropic Murine Leukemia Virus–related Virus Detected in Fibromyalgia Patients

Testing Strategies for Detection of Xenotropic Murine Leukemia Virus-Related Virus Infection

Xenotropic murine leukemia virus-related virus (XMRV) is a newly identified gamma retrovirus and may be associated with prostate cancer- (PC) and chronic fatigue syndrome (CFS). Since its identification in 2006 and detection of polytropic murine lenkemia virus (MLV)-like sequences in CFS patients in 2010, several test methods including nucleic acid testing methods and serological assays have been developed for detection of XMRV and/or MLV-like sequences. However, these research assays have not yet been validated and evaluated due to the lack of well-characterized reference materials. Mouse DNA contamination should be carefully checked when testing human specimens in order to avoid false-positive detection of XMRV or MLV-like sequences.

No Evidence for XMRV in German CFS and MS Patients with Fatigue Despite the Ability of the Virus to Infect Human Blood Cells In Vitro

BackgroundXenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms.MethodologyBlood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production.ConclusionNone of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.

Contamination of clinical specimens with MLV-encoding nucleic acids: implications for XMRV and other candidate human retroviruses

Efforts to assess the prevalence of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome have relied heavily on PCR-based testing of clinical samples and have yielded widely divergent findings. This week in Retrovirology, reports from four independent research groups illustrate the extreme care needed to exclude DNA or RNA contamination in PCR analyses of XMRV. In addition, phylogenetic evidence suggesting that previously-published XMRV sequences originated from a commonly-used prostate carcinoma cell line (22Rv1) is presented. These findings raise important questions regarding the provenance of XMRV and its potential connection to human disease.

No evidence for XMRV association in pediatric idiopathic diseases in France

Retroviruses have been linked to a variety of diseases such as neoplastic and immunodeficiency disorders and neurologic and respiratory diseases. Recently, a novel infectious human retrovirus, the xenotropic murine leukemia virus-related virus (XMRV), has been identified in cohorts of patients with either a familial type of prostate cancer or chronic fatigue syndrome. The apparent unrelatedness of these diseases raised the question of the potential involvement of XMRV in other diseases.Here, we investigated the presence of XMRV in a selection of pediatric idiopathic infectious diseases with symptoms that are suggestive of a retroviral infection, as well as in children with respiratory diseases and in adult patients with spondyloarthritis (SpA). Using a XMRV env-nested PCR, we screened 72 DNA samples obtained from 62 children hospitalized in the Montpellier university hospital (France) for hematological, neurological or inflammatory pathologies, 80 DNA samples from nasopharyngeal aspirates from children with respiratory diseases and 19 DNA samples from SpA. None of the samples tested was positive for XMRV or MLV-like env sequences, indicating that XMRV is not involved in these pathologies.