XMRV not detected in seminal plasma

How XMRV, the new human retrovirus associated with prostate cancer and chronic fatigue syndrome, might be transmitted among humans is unknown. The finding that the virus can be detected in prostate cancer cells, and in prostatic secretions of men with prostate cancer suggests that it could be sexually transmitted. To address this question, the presence of XMRV in seminal plasma of men with HIV-1 was examined. Although the virus was not detected in 93 samples from 54 HIV-1 infected men, the study provides little information on possible transmission mechanisms of XMRV.

This study involved two groups of HIV-1 infected men from the Netherlands: 29 who have sex with men, and 25 heterosexual men. The rationale for examining HIV-1 infected men for XMRV was that “they have a higher chance of contracting sexually transmitted pathogens than non-HIV-1 infected men”. For 39 men a second sample was also available from another time point, bringing the total samples to 93.

To detect XMRV, semen samples were diluted 1:1 with buffer and centrifuged to remove cells, yielding seminal plasma. Total nucleic acid was then extracted and subjected to reverse-transcription and then polymerase chain reaction. This procedure assays for the presence of XMRV viral RNA. At the same time, the samples were also tested for the presence of HIV-1 RNA. The positive control for XMRV was total nucleic acid extracted from a prostate cancer cell line known to produce viral RNA.

The results show that HIV-1 was detected in 25% of the seminal plasma samples, while none contained XMRV nucleic acid. The authors conclude:

Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.

In my opinion, these conclusions are not supported by the data obtained in this study. Here are my reasons:

  • The semen samples were subjected to centrifugation, which removes all cells, including spermatozoa, epithelial cells, and lymphocytes. Such cells could harbor virions.
  • The study was designed only to search for XMRV virions or viral RNA, not proviral DNA, which is integrated into cellular DNA.
  • No attempt was made to determine if the 54 men were infected with XMRV. This could have been done by taking blood samples and co-culturing them with LNCaP cells, then performing PCR. If none of the men were infected with the virus, then absence of the virus in their semen is meaningless.

To determine if XMRV could be transmitted in semen, I would obtain semen samples from patients known to be infected with the virus. Then I would co-culture total semen and seminal plasma with LNCaP cells to amplify any virus present, followed by PCR to detect either virions or proviral DNA. I realize it may be difficult to conduct the study in this way, but I don’t see the value of doing it any other way.

Marion Cornelissen, Fokla Zorgdrager, Petra Blom, Suzanne Jurriaans, Sjoerd Repping, Elisabeth van Leeuwen, Margreet Bakker, Ben Berkhout, & Antoinette C. van der Kuyl (2010). Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands PLoS One : 10.1371/journal.pone.0012040

TWiV 94: XMRV with Dr. Ila Singh

Hosts: Vincent Racaniello, Alan DoveRich Condit, and Ila Singh

On episode #94 of the podcast This Week in Virology, Vincent, Alan, and Rich speak with Ila Singh about the new human retrovirus XMRV, and how her laboratory is studying its association with prostate cancer and chronic fatigue syndrome.

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XMRV, prostate cancer, and chronic fatigue syndrome

Robert H. Silverman, one of the authors on the study implicating the new human retrovirus XMRV as an etiologic agent of chronic fatigue syndrome, has written an excellent review article on the current status of research on the virus. The article is behind a paywall at Nature Reviews Urology, so I’ll provide a summary of the salient points.

The article begins with a description of how XMRV DNA was isolated from surgically removed prostate tumor tissue. Sequence analysis of three strains showed that the virus is most closely related to xenotropic and polytropic murine leukemia viruses and hence was named xenotropic murine leukemia virus-related virus, or XMRV. Five lines of evidence indicate that XMRV is not a laboratory contaminant:

  • XMRV was detected in RNA isolated from human prostate tissue
  • Mouse sequences were not detected in the human prostate tissues
  • Infections were mainly found in tissues from humans with an alteration in the protein RNAse L
  • Slightly different viral sequences (polymorphisms) were identified in isolates from different patients
  • Both viral RNA and viral proteins were detected in prostate tissues

The article continues with a summary of  subsequent studies in which XMRV has or has not been detected in prostate cancer, then moves to the role of XMRV in CFS. Silverman concludes that “the scientific literature shows that XMRV was detected in the majority, but not all, prostate cancer studies, albeit at different rates, while XMRV was found in CFS in only one study of four published to date.” He offers the the following explanations for the difference in detection of XMRV in prostate cancer and CFS:

  • Viral contamination from mouse sources. This cannot explain all positive findings in different laboratories using different experimental techniques.
  • Geographical differences in the distribution of XMRV could account for some of the differences.
  • Sequence differences could lead to failure to detect viral DNA by polymerase chain reaction.
  • There are no standardized, sensitive methods of detection, and no widely available positive control samples.

The remainder of the article entails speculation on how XMRV might cause prostate cancer; the likely origin of the virus from a rodent virus; and antiviral drugs that are known to inhibit replication of the virus. It ends with the suggestion to test for the presence of XMRV in porcine tissues used for human transplantation, and in the blood supply, to avoid additional infections.

Silverman concludes:

Although other retroviruses of the same genus as XMRV (gammaretroviruses) cause cancer and neurological disease in animals, whether XMRV is a cause of either prostate cancer or CFS remains unknown.

Robert H. Silverman, Carvell Nguyen, Christopher J. Weight & Eric A. Klein. The human retrovirus XMRV in prostate cancer and chronic fatigue syndrome. Nature Reviews Urology doi: 10.1038/nrurol.2010.77.

XMRV in human respiratory tract

An important question about the retrovirus XMRV, which has been implicated in prostate cancer and chronic fatigue syndrome, is where the virus replicates in humans. Such information would provide clues about how infection might be transmitted. To date the virus has been detected in malignant prostate cells and in the peripheral blood mononuclear cells and plasma of patients with CFS. A new study reveals that XMRV is present in respiratory secretions.

Polymerase chain reaction was used to detect XMRV in 267 respiratory samples taken from German patients. One group comprised sputum and nasal swab specimens from 75 travelers from Asia who had respiratory tract infections. The second group consisted of 31 bronchoalveolar lavage samples from patients with chronic obstructive pulmonary disease, while samples from the third group were from 161 immunosuppressed patients with severe respiratory tract infections. The study included 62 healthy controls. It should be noted that none of the patients had been diagnosed with CFS.

XMRV sequences were detected in 3 of 75 samples (2.3%) in group 1, 1 of 31 samples (3.2%) in group 2, and 16/161 (9.9%) in group 3. Six of the XMRV-positive samples in the second group also contained rhinovirus, adenovirus, or pathogenic fungi. The higher rate of detection of XMRV and other microbes in immunosuppressed individuals is not unexpected. The control group contained 2 of 62 samples (3.2%) positive for XMRV.

The presence of XMRV in PBMCs and plasma suggests a blood-borne route of transmission of the virus: transfusions, health care associated needle sticks, and intravenous drug use. Does finding XMRV in the respiratory tract prove that the virus can be transmitted by the respiratory route? No, not until we have other information, including the level of virus in respiratory secretions, and the infectivity of XMRV. In this context it is interesting to note that it was not possible to isolate infectious XMRV from the respiratory tract of the German patients.

Reviewing the transmission of another human retrovirus, HIV-1, is instructive in understanding the pathogenesis of XMRV infection. The main modes of transmission of HIV-1 are sexual, parenteral, and from mother to infant. These routes of transmission are consistent with levels of infectious virus in body fluids (shown in this table). Viral RNA can be detected at several levels of the respiratory tract, but respiratory secretions rarely transmit HIV.

FIsher, N., Schulz, C., Stieler, K., Hohn, O., Lange, C., Drosten, C., & Aepfelbacher, M. (2010). Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract Emerg. Inf. Dis. : 10.3201/eid1606.100066

XMRV at Cold Spring Harbor

Cold Spring Harbor Laboratory (CSHL) is a private, non-profit institution located in the eponymous town on Long Island, New York. Over 400 scientists work there on a wide range of biological problems, including cancer, neurobiology, plant genetics, and genomics. CSHL has a storied research history, having hosted nine Nobel Laureates. But it is also well known for its world class scientific conferences. The first of these was the Cold Spring Harbor Laboratory Symposium on Quantitative Biology, which was held in 1934. Another well known event is the Phage Course, founded by Salvador Luria and Max Delbrück in 1948. There are now over 24 meetings held annually. One of these is the meeting on retroviruses, which will begin on 24 May 2010. Below is a list of the presentations about XMRV, the new retrovirus implicated in prostate cancer and chronic fatigue syndrome. The author presenting each study can be found at the meeting website.

  • Failure to detect XMRV in human prostate tumors
  • Development of a multiplex serological assay to detect XMRV antibodies
  • Characterization of cellular determinants required for infection of XMRV, a novel retrovirus associated with human familial prostate cancer
  • Screening mouse genomes For XMRV-Like Elements
  • Development of highly sensitive assays for the detection of XMRV nucleic acids in clinical samples
  • Compounds that inhibit replication of XMRV, a virus implicated in prostate cancer and chronic fatigue syndrome
  • Investigation of XMRV as a human pathogen
  • Investigations into XMLV-related virus infection
  • XMRV is not detected in Quebec patients with chronic fatigue syndrome
  • Wild-derived mouse strain (Mus pahari) as a small animal model for XMRV infection
  • XMRV tropism in hematopoietic cells
  • Evidence for sequence variation in XMRV
  • The human retrovirus XMRV produces rare transformation events in cell culture but does not have direct transforming activity
  • The XMRV is inhibited by APOBEC3 proteins and anti-HIV-1 drugs
  • Immune responses in XMRV-infected rhesus macaques—Serological markers of XMRV infection
  • XMRV Is inhibited by interferon independently of RNase L or Tetherin
  • Comparison of XMRV infections in humans and rhesus macaques
  • Susceptibility of XMRV to antiretroviral inhibitors
  • Integration site analysis in XMRV-positive prostate cancers
  • Xpr1 is necessary but not sufficient for XMRV entry
  • Effects of interferon regulated proteins, RNase L and APOBEC3G, on XMRV replication

The retrovirus community has clearly embraced XMRV, a virus discovered just four years ago. This high level of activity means that there will be many papers on XMRV in the scientific literature in the next year. I’m looking forward to discussing them with the readers of virology blog.

Inhibition of XMRV by a weapon of mass deamination

deaminationCtoUAll mammalian genomes contain genes encoding Apobec proteins. Several members of this protein family (the name stands for apolipoprotein B mRNA editing complex) are induced by interferon and are intrinsic antiretroviral proteins. Apobec proteins inhibit the replication of XMRV, a new human retrovirus associated with prostate cancer and chronic fatigue syndrome.

During retroviral replication, Apobec proteins are packaged into newly synthesized retrovirus particles (illustrated). apobec_virionThey exert their antiviral effect when Apobec-containing virions infect a new cell. As the viral reverse transcriptase begins to copy viral RNA into DNA, Apobec removes an amine group from cytosines in single stranded DNA, a process called deamination.  The consequence of deamination is that cytosine is changed to uracil. Uracil-containing DNA may be attacked by uracil DNA glycosidase, which removes the base and makes the DNA susceptible to degradation. If deaminated DNA is copied to form a double-stranded molecule, the new Us pair with As. In other words, deamination leads to a G-to-A mutation in the viral DNA. The highly mutated DNA cannot encode viable viruses, and the infection is terminated. For this reason one retrovirologist has called Apobec a WMD – a weapon of mass deamination.

Apobec is lethal for retroviruses that incorporate the enzyme into their virions. Human immunodeficiency virus-1 counters this defense by producing the Vif protein, which binds to Apobec and promotes its degradation by cellular enzymes.

XMRV does not encode a Vif protein and should be susceptible to inhibition by Apobec proteins. To answer this question, XMRV virions were produced in cells in the presence of different Apobec proteins. The deaminases were incorporated into virions, where they resulted in G-to-A hypermutation and inhibition of viral infectivity.

Could the presence of Apobec determine which human tissues are infected with XMRV? The virus replicates very well in a prostate cancer cell line, LNCaP, which produces reduced levels of Apobec proteins. Whether Apobec could regulate XMRV replication in the prostate is not known because expression of the protein in normal or malignant prostate tissues has not been studied. A conundrum which requires further investigation concerns the isolation of XMRV from CD4+ T and B cells, which are known to synthesize Apobec proteins. How XMRV might evade Apobec inhibition in these cells remains unexplained.

Paprotka, T., Venkatachari, N., Chaipan, C., Burdick, R., Delviks-Frankenberry, K., Hu, W., & Pathak, V. (2010). Inhibition of Xenotropic Murine Leukemia Virus-Related Virus by APOBEC3 Proteins and Antiviral Drugs Journal of Virology DOI: 10.1128/JVI.00134-10

Groom, H., Yap, M., Galao, R., Neil, S., & Bishop, K. (2010). Susceptibility of xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors Proceedings of the National Academy of Sciences, 107 (11), 5166-5171 DOI: 10.1073/pnas.0913650107

XMRV not found in 170 additional UK chronic fatigue syndrome patients

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 68: Ode to a plaque

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich are enthralled by movies of vaccinia virus plaque formation, then consider how repulsion of superinfection virions leads to rapid virus spread, and a therapeutic prostate cancer vaccine.

This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S.

Win a free Drobo S! Contest rules here.

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Rich Foundations of Virology – PowerPoint by Frederick A. Murphy (bio/interview pdf)
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XMRV infection is enhanced by prostatic protein fragments

amyloid_temFragments of an abundant protein produced by the prostate form amyloid fibrils that enhance infection of cells by human immunodeficiency virus type 1. These fibrils, called semen-derived enhancer of virus infection (SEVI), have been found to boost infection of prostate cells by the retrovirus XMRV. Is this evidence that XMRV causes prostate cancer?

Because most HIV-1 infections are a consequence of genital exposure to semen of virus-infected men, seminal fluid was screened for peptides or proteins that enhance viral infectivity. Peptides (34 – 40 amino acids in length) derived from prostatic acidic phosphates, a common protein found in semen, were found to dramatically enhance HIV-1 infection of cultured cells. These peptide fragments form amyloid fibrils which bind both virions and cells, thereby promoting virus attachment and stimulating infectivity. The prostatic amyloid fibrils, called SEVI, are found at high levels in semen, which also stimulates HIV infection. SEVI is believed to play an important role in sexual transmission of HIV. Inhibitors of the stimulatory effect of SEVI on HIV infection, such as surfen, may have therapeutic value.

Xenotropic murine leukemia virus-related virus (XMRV) has been detected in prostate cancer tissues and is therefore a candidate tumor virus. XMRV, which has also been implicated in chronic fatigue syndrome, was first isolated from prostate tissue. Therefore it made perfect sense to determine whether SEVI, which originates from the prostate gland, enhances XMRV infection.

The observation that SEVI enhances XMRV infection is consistent with the possibility that the virus is sexually transmitted. Men with a history of prostatitis or sexually transmitted infections appear to have a higher risk of acquiring prostate cancer. However, the effect of a prostate-derived peptide on XMRV infection might be coincidental: the amyloid fibrils could stimulate infection by other viruses, as noted by the virologists who discovered SEVI:

…the capability to promote the interaction between virions and the cell surface is independent of the viral Env glycoprotein and hence not restricted to retroviruses. Thus, further studies on the role of amyloids in the transmission and pathogenesis of enveloped viruses are highly warranted.

About 30 human diseases, including Alzheimer’s, are associated with deposits of amyloid. Bacterial and fungal infections can also lead to formation of amyloid fibrils – which could explain why sexually transmitted diseases increase the likelihood of acquiring prostate cancer. The ability of these fibrils to enhance infection with different viruses should be examined. It’s possible that different amyloid fibrils are a new general risk factor for certain viral infections.

Hong, S., Klein, E., Das Gupta, J., Hanke, K., Weight, C., Nguyen, C., Gaughan, C., Kim, K., Bannert, N., Kirchhoff, F., Munch, J., & Silverman, R. (2009). Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer Journal of Virology, 83 (14), 6995-7003 DOI: 10.1128/JVI.00268-09

TWiV 64: Ten virology stories of 2009

3D_InfluenzaHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich discuss ten compelling virology stories of 2009.

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Ten virology stories of 2009:

  1. Pandemic influenza: Swine-origin H1N1 virus (TWiV 36)
  2. XMRV, prostate cancer, and chronic fatigue syndrome (TWiV 50, 55)
  3. AIDS vaccine ‘success’ (TWiV 51)
  4. Colony collapse disorder (TWiV 46, 49)
  5. AIDS-like disease in wild chimps (TWiV 45)
  6. Diverse viral community in Antarctic lake (TWiV 58)
  7. Polyomavirus seroepidemiology in humans (TWiV 26)
  8. Poxvirus threatens UK red squirrels (TWiV 63)
  9. Polio spreads from Nigeria (TWiV 29)
  10. How mosquitoes survive Dengue virus infection (TWiV 21)

Picture book on viruses for kids (Thanks Soraia!)

Weekly Science Picks
Rich Surely You’re Joking, Mr. Feynman! by Richard P. Feynman, Ralph Leighton, Edward Hutchings, and Albert R. Hibbs
Alan Spaceweather.com
Vincent The Art and Politics of Science by Harold Varmus

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