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chronic fatigue syndrome

XMRV not found in 170 additional UK chronic fatigue syndrome patients

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xmrv_neutralizationA new retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), first identified in tumor tissue of individuals with prostate cancer, was subsequently found in 68 of 101 US patients with chronic fatigue syndrome (CFS). XMRV was not detected in blood samples of 186 confirmed CFS patients in the United Kingdom. A second independent study in the UK (pdf) has also failed to reveal XMRV in CFS patients.

The subjects of this study were confirmed CFS patients from St George’s University of London, Barts and the London Hospital Trust, and Glasgow Caledonian University. A total of 170 serum samples from CFS patients and 395 controls were used. A polymerase chain reaction assay was devised that could detect as little as 16 copies of proviral XMRV DNA (viral DNA integrated into human chromosomal DNA). No XMRV sequences were detected in 142 CFS samples and 157 controls.

A second method was then used to search for evidence of XMRV: the patient serum samples were examined for the presence of antibodies that could block infection of cells with the virus. Cells were infected with XMRV in the presence of serum from CFS patients or control patients. Included were sera known to block XMRV infection to ensure that the assay functioned normally. None of 142 CFS samples contained antibodies that could block XMRV infection of cells. In contrast, 22 samples out of 157 controls (14%) were identified that contained neutralizing activity. One of 28 CFS serum samples from a separate cohort was found to contain XMRV neutralizing activity; none of the 12 control sera could block XMRV infection.

These results could be interpreted to mean that XMRV infection occurs in the general population, confirming the observations of the first US study. However, the sera from the second UK study also blocked the infectivity of viruses other than XMRV, including those containing envelope proteins from vesicular stomatitis virus. The authors believe that the neutralizing activity in the control sera is not specific for XMRV. These antibodies were probably induced by infection with another virus.

The results obtained with these samples do not provide evidence for an association of XMRV infection and CFS. This does not eliminate a role for XMRV in CFS. As the authors write:

The publication of these results has promoted much discussion and controversy amongst CFS researchers and patients alike, and has highlighted the need for additional investigations in this area. Following the findings reported here, it would seem a prudent next step for subsequent studies to compare samples and protocols between different laboratories around the world.

It’s time to put aside arguments over the competence of laboratories to carry out polymerase chain reaction and work towards understanding the role of XMRV in human disease. The three laboratories who have published their findings on XMRV in humans should exchange their samples to confirm the findings. Compelling answers will only come from far more extensive global studies of the prevalence of XMRV in CFS and control populations are clearly needed.

Harriet C T Groom, Virginie C Boucherit, Kerry Makinson, Edward Randal, Sarah Baptista, Suzanne Hagan, John W Gow, Frank M Mattes, Judith Breuer, Jonathan R Kerr, Jonathan P Stoye, & Kate N Bishop (2010). Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome Retrovirology : 10.1186/1742-4690-7-10

TWiV 65: Matt’s bats

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Hosts: Vincent Racaniello, Alan Dove, and Matthew Frieman

Vincent, Alan, and Matt discuss a project to study the RNA virome of Northeastern American bats, failure to detect XMRV in UK chronic fatigue syndrome patients, and DNA of bornavirus, an RNA virus, in mammalian genomes.

This episode is sponsored by Data Robotics Inc. To receive $50 off a Drobo or $100 off a Drobo S, visit drobostore.com and use the promotion code VINCENT.

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XMRV not detected in UK chronic fatigue syndrome patients

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xmrv_pcrA new retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), first identified in tumor tissue of individuals with prostate cancer, was subsequently found in 68 of 101 US patients with chronic fatigue syndrome (CFS). This observation raised the possibility that XMRV is the etiologic agent of CFS. An important question is whether XMRV is associated with CFS in other parts of the world. For some CFS patients in the UK the answer appears to be no.

The subjects of this study were 186 confirmed CFS patients who had been referred to the CFS clinic at King’s College Hospital, London. DNA was prepared from blood samples and subjected to polymerase chain reaction using primers that anneal to an XMRV-specific sequence, and to a sequence conserved among murine leukemia viruses. To demonstrate that the amplification worked, a positive control reaction was done using XMRV DNA. The reaction products were fractionated by electrophoresis on an agarose gel, and the DNAs were visualized by staining with a dye. As shown in the figure, positive control samples containing XMRV DNA produced DNAs of the expected sizes (lanes 10, 11). None of the 186 test DNAs from CFS patients produced a PCR product with either the XMRV or murine leukemia virus primers. Some of these negative samples are shown in lanes 1-8 of the figure.

The authors of this study suggest that they were more careful at avoiding contamination than the group which previously identified XMRV in American CFS patients:

…the PCR operator was blinded to the provenance of the DNA samples. In fact, with the exception of the PCR controls, all 186 DNA test samples originated from CFS patients. Care was taken to grow the XMRV plasmid in a laboratory in which no MLV had been cultured and no MLV vectors used and the PCR was carried out in a CPA-accredited Molecular Diagnostics Unit which processes only human tissue. Multiple (six) water (negative) controls were included in every run to detect low level contamination and a PCR to amplify a sequence that is conserved in most murine leukaemia viruses was included in order to expose any circulating MLV contamination and to detect any variant of XMRV that might be circulating in the UK CFS population.

But they also acknowledge that there might be population differences in XMRV distribution:

Based on our molecular data, we do not share the conviction that XMRV may be a contributory factor in the pathogenesis of CFS, at least in the U.K.

These results are surely a disappointment to CFS sufferers who believe that XMRV is the etiologic agent of the disease. But they reveal the dangers of making conclusions about disease etiology based on the findings of limited studies. As I wrote previously, while the presence of XMRV in 67% of CFS samples seems impressive, it could be misleading. For example, the samples could be from regions where XMRV infection is common.

In light of these new findings, it is informative to recall that the nucleic acid of another retrovirus, human T-lymphotropic virus type ll, was previously amplified from blood cells of 23 of 30 CFS patients, but not from healthy patients. However this observation was not confirmed in subsequent studies of CFS patients. In addition, there is no evidence for the presence of other retroviruses in CFS patients, including HIV-1, bovine and feline leukemia viruses, simian T lymphotropic virus type l, foamy virus, and simian retrovirus.

It is possible that XMRV is involved in CFS but only in certain parts of the world. More extensive studies such as the one reported here must be done worldwide to clarify the role of XMRV in CFS, and to determine whether other infectious agents are involved.

Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, & Cleare A (2010). Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PloS one, 5 (1) PMID: 20066031

Futures in Biotech 50: More biotech stories

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futures-in-biotechI joined host Marc Pelletier and George Farr, Justin Sanchez, and Dave Brodbeck for a discussion on recent big stories in bioscience. Topics included erasing memory, controlling neurons with light, the role of the new virus XMRV in prostate cancer and chronic fatigue syndrome, and prions as genetic elements in yeast.

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Download Futures in Biotech 50 or subscribe to the podcast in iTunes.

AZT inhibits XMRV

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aztXenotropic murine leukemia virus related virus (XMRV) has been implicated in prostate cancer and chronic fatigue syndrome (CFS). Because XMRV is a retrovirus, it has been suggested that it might be susceptible to some of the many drugs available for treatment of AIDS. Of ten licensed compounds evaluated for activity against XMRV, just one, AZT (azidothymidine), was found to inhibit viral replication.

Compounds used to treat HIV-1 infection fall into distinct classes: protease inhibitors (Ritonavir, Saquinavir, or Indinavir), nucleoside reverse transcriptase inhibitors (NRTI, AZT, 3TC, Tenofovir, D4T), non-nucleoside reverse transcriptase inhibitors (NNRTI, Efavirenz, Nevirapine), integrase inhibitors (118-D-24), and fusion inhibitors (Maraviroc). None of the HIV-1 protease inhibitors, NNRTI, or integrase inhibitors blocked XMRV replication.  Of the NRTIs, only AZT significantly inhibited viral replication. Fusion inhibitors were not examined in this study.

AZT was the first drug licensed to treat AIDS. It is phosphorylated to the active form by cellular enzymes. Phosphorylated AZT is an inhibitor of viral reverse transcriptase because it acts as a chain terminator when incorporated into DNA:

azt_mechanism

Because AZT has a N3 (azido) group on the ribose instead of a hydrogen, the next base cannot be added to the DNA chain and synthesis stops.

The relative selectivity of this drug depends on the fact that reverse transcription takes place in the cytoplasm, where the drug appears first and in the highest concentration. But the presence of AZT monophosphate causes depletion of the intracellular pool of ribosylthymine 5′-triphosphate (TTP). Therefore AZT has substantial side effects which include muscle wasting, nausea, and severe headaches. AZT treatment can also damage bone marrow, which requires multiple transfusions of red blood cells. The drug was used extensively because there was no alternative until other antivirals were developed.

AZT can be taken orally but it is degraded rapidly by liver enzymes. Patients must take the drug two or three times a day to maintain an effective antiviral concentration. The drug is modestly effective in infected adults, leading to a transient increase in CD4+ T-cell counts.

Much effort has been devoted to discovering alternatives to AZT, and several nucleoside analogs that have therapeutic value, such as 3TC, are available. However 3TC does not inhibit XMRV replication.

It is not known if treatment with AZT will effect either prostate cancer or CFS. If prostate cancer is triggered when XMRV inserts into chromosomal DNA, then the drug will not likely block progression of the disease because the drug does not eliminate infected cells. Whether reduction of viral loads by AZT treatment has a positive therapeutic outcome remains to be determined. Because AZT is approved for use in humans, such studies can proceed immediately, without the need for extensive toxicity studies in animals.

Sakuma R, Sakuma T, Ohmine S, Silverman RH, & Ikeda Y (2009). Xenotropic murine leukemia virus-related virus is susceptible to AZT. Virology PMID: 19959199

Raltegravir inhibits murine leukemia virus: implications for chronic fatigue syndrome?

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RaltegravirThe finding that a retrovirus, XMRV, is associated with chronic fatigue syndrome has lead to the suggestion that the disease might be treated with some of the antiviral drugs used to treat AIDS. The integrase inhibitor Raltegravir has been found to block the replication of murine leukemia virus, which is highly related to XMRV. But the drug exacerbates autoimmune disease in mice which might rule out its use in treating CFS.

Retroviruses such as XMVR and HIV-1 have genomes composed of single-stranded RNA. This nucleic acid is converted to a DNA copy in infected cells by the viral enzyme reverse transcriptase. The double-stranded viral DNA is then integrated into the chromosomal DNA of the host cell, a process accomplished by an viral enzyme called integrase (illustrated).

retroviral_integration

Raltegravir (pictured above left) is an inhibitor of HIV-1 integrase that was approved for use in humans in 2007. The drug blocks the integration of viral DNA into the host genome and therefore inhibits viral replication.

The mouse retrovirus murine leukemia virus (MLV) has been linked to the development of spontaneous autoimmune disease. The mechanism by which the virus induces this disease is not known, but stimulation of innate immune responses by viral DNA might be involved.

Raltegravir also inhibits integration of MLV DNA into the murine genome. When mice with autoimmune disease were treated with raltegravir, they succumbed to autoimmune disease a month earlier than untreated animals. Mice without the disease were not affected by the antiviral drug. The authors speculate that by inhibiting viral DNA integration, raltegravir increases the amount of unintegrated viral DNA, elevating innate responses and exacerbating autoimmunity.

It’s not known if raltegravir is active against XMRV, the retrovirus associated with chronic fatigue syndrome. Given the similarity between the genomes of MLV and XMRV it seems likely that the drug will inhibit the virus. If the ability of raltegravir to treat CFS is tested in clinical trials, it will be important to carefully monitor treated patients for signs of autoimmunity. CFS has an autoimmune component which could worsen with raltegrivir treatment.

An obvious question is whether raltegrivir induces autoimmunity in AIDS patients. I’m not aware of any such reports, which is probably not surprising given the fact that HIV-1 infection leads to immunosuppression.

CFS sufferers should not despair: other antiretroviral drugs, including chain terminators such as AZT, do not allow the accumulation of unintegrated viral DNA. These compounds might be useful for treating the disease.

G.B. Beck-Engeser, D. Eilat, T. Harrer, H.-M. Jack, M. Wabl (2009). Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir Proceedings of the National Academy of Sciences : 10.1073/pnas.0908074106