18th Conference on Retroviruses and Opportunistic Infections

February 27 – March 2, 2011

XMRV-RELATED ABSTRACTS
(FROM THE CROI WEBSITE)

PRESENTATIONS DURING SESSION: “XMRV: NEW FINDINGS & CONTROVERSIES”

Title: Development of a GFP-indicator Cell Line for the Detection of XMRV
Authors and Affiliations: KyeongEun Lee, F Ruscetti, P Lloyd, A Rein, G Fanning-Heidecker, and V KewalRamani NCI-Frederick, MD, US

Background: HIV titer can be estimated using indicator cell lines within days of infection. HIV indicator cells rely on production of tat to transactivate expression of a reporter gene under the control of HIV LTR sequences. Simple retroviruses typically do not encode transcriptional transactivators. For simple retroviruses that lack transformational or cytopathic activity, their titers are measured by endpoint dilution and assaying for virus proliferation after weeks of cell culture. Replication-dependent vectors have been used to monitor mobilization by retrotransposable elements or the replication of retroviruses. Here we describe an indicator cell line for the detection of infectious xenotropic murine leukemia virus-related virus (XMRV) that relies on the propagation of a vector, which leads to expression of a GFP reporter.

Methods: We constructed a murine leukemia virus vector encoding puromycin resistance and a CMV enhancer/promoter driven GFP reporter gene whose transcription was antisense to the vector mRNA. The GFP reporter sequence (iGFP) was interrupted by an intron placed in the sense direction relative to the vector. The prostate cell line, LNCaP, was stably transfected with the above construct and drug-resistant clones were isolated. GFP was detected by fluorescence microscopy or FACS analysis.

Results: Several LNCaP-iGFP cell clones displaying sensitivity to XMRV infection after endpoint dilution were isolated and designated Detectors of Exogenous Retroviral Sequence Elements (DERSE) cells. GFP signal could be detected within 3 days of infection, with the number of GFP+ cells increasing over time. GFP signal after virus inoculation was dose-dependent and could be impaired by heat inactivation or the addition of zidovudine to cultures at the time of infection.

Conclusions: In principle, DERSE cells should also detect other gammaretroviruses capable of infecting human cell lines. Because this indicator cell system utilizes GFP as a reporter, infection can be monitored in live cultures by fluorescence microscopy. DERSE cells provide a facile assay to assess antiviral or antibody mediated neutralization of XMRV, and should be useful in assessing the presence of infectious XMRV in patient samples.
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Title: XMRV Induces a Nonproductive Infection in Human Lymphoid Tissue
Authors and Affiliations: Marta Curriu1, J Carrillo1, M Massanella1, E Garcia1, B Clotet1, C Carrato2, J Blanco1, and C Cabrera1 1Fndn irsiCaixa, Badalona, Spain and 2Hosp Univ Germans Trias i Pujol, Barcelona, Spain

Background: Xenotropic murine leukemia virus-related virus (XMRV) has been associated with prostate cancer and chronic fatigue syndrome. In humans the virus has been found in a variety of cell types, including T and B cells, and in rhesus macaques there is evidence for viral replication in lymphoid organs, suggesting that lymphocytes are a target for XMRV. Histocultures of tonsils support productive infection with various viruses, including HIV and human herpesvirus 6. In this study, ex vivo lymphoid tissue was used to investigate the pathogenic mechanisms of XMRV.

Methods: Tonsils from 2 healthy individuals undergoing tonsillectomy were collected and cultured in small pieces (2 mm3) over gelfoam soaked in RPMI medium. Tissue blocks were left uninfected or infected with XMRV stock obtained from a 22Rv1 cell supernatant. Culture medium was replaced every 3 days. After 14 days in culture, both tissues were homogenized and cells were isolated. Viral infection was evaluated at different times in the cells migrating out the tissue and at day 14 in tissue cells by PCR, analyzing viral DNA. In addition, tissue cells were immunophenotyped by flow cytometry and the presence of envelope protein (env) was analyzed by Western blot (WB) using an antibody to SFFV env that reacts with all poly- and xenotropic murine leukemia virus.

Results: Seven days post-infection cells migrating out the tissue were positive for XMRV DNA. After 14 days of culture, tissue cells were also positive, confirming that XMRV infected human tonsil tissue in the absence of exogenous stimulation. Despite the presence of XMRV DNA, infection does not seem to be productive since tissue lysates exhibited undetectable expression of XMRV env proteins by WB. Uninfected and infected tissues showed similar percentages of T and B cells. XMRV infection did not modify the percentage of CD3 (76 and 75% in XMRV+ and XMRV– tissue, respectively), CD4 (53% vs 52%), CD8 (39% vs 40%), or CD19 cells (3% vs 1%). A deeper analysis of T cell subsets showed that XMRV infection did not modify the naïve/memory cell ratio, or immune activation markers, as evaluated by the expression of HLA-DR and CD38.

Conclusions: Our data show that XMRV could be integrated into the human lymphoid tissue cells although this process does not culminate in an explicit productive infection. In addition, this infection did not result in changes of T or B cells nor an immune activation, suggesting that lymphoid tissue could be a latent tissue reservoir in XMRV infection.
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Title: Presence of XMRV Sequences in B Cells Are Restricted by APOBEC
Authors and Affiliations: Jorge Carrillo1, J Blanco1, E Garcia1, J Arreal2, B Clotet1, and C Cabrera1 1Fndn irsiCaixa, Barcelona, Spain and 2Hosp Univ Germans Trias i Pujol, Barcelona, Spain

Background: A link between xenotropic murine leukemia virus-related virus (XMRV) infection and different human diseases, such as chronic fatigue syndrome (CFS) and prostate cancer, has been recently established. Given that this retrovirus can infect different tissues and cell types both in vivo and in vitro, including cells from the immune system, the identification of the cellular compartment(s) where XMRV can establish a reservoir may be useful to understand the pathology associated with this virus.

Methods: In order to determine the presence of XMRV in B lymphocytes, we have screened EBV-transformed B-cell lines available in our laboratory. The origin of these cell lines include CFS patients (n = 11; fulfilling both Fukuda and Canadian criteria), HIV+ individuals (n = 4), prostate cancer patients (n = 1), and healthy donors (n = 5). DNA was extracted from cellular dry pellets and several XMRV genes were amplified by either real-time PCR (pol) or nested PCR (gag and env).

Results: We detected 7 positive samples from 14 individuals tested by RT-PCR (4 CFS, 2 donors, and 1 HIV+ individual). env amplification identified 4 positive samples out of 21 individuals tested (3 CFS-affected individuals and 1 healthy donor), whereas gag amplification showed only 3 positive samples (1 CFS-affected individual, 1 healthy donor, and one HIV+ patient). To confirm the presence of XMRV we performed sequence analyses of gag and env amplicons. The 3 gag sequences available showed a 100% homology with XMRV gag published sequences, including the XMRV characteristic 24nt deletion which is not found in any known exogenous murine leukemia virus. Furthermore, env sequences were also homologous to previously described XMRV sequences, showing none or low variability. Interestingly, most of the observed changes corresponded to multiple G to A mutations that were accumulated in 1 positive sample, resulting in a truncated env protein and suggesting that APOBEC-related restrictions operate in vivo during XMRV infection.

Conclusions: Despite the discrepancies observed in the different PCR approaches, our data provide evidence that EBV-transformed B-cell lines harbor XMRV-specific sequences, although the establishment of this infection could be modulated by the innate restriction factor APOBEC 3G. Our data suggest that, in vivo, B cells may represent a reservoir for XMRV contributing to its potential pathogenesis.
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Title: Single Copy and Single Genome Sequencing Assays to Detect XMRV in Human Blood Products
Authors and Affiliations: Mary Kearney 1, A Wiegand1, J Spindler1, F Maldarelli1, J Mellors2, J Coffin3, and Blood XMRV Scientific Research Working Group 1HIV Drug Resistance Program, NCI-Frederick, MD, US; 2University of Pittsburgh, PA, US; and 3Tufts University, Boston, MA, US

Background: The human retrovirus xenotropic murine leukemia virus-related virus (XMRV) was recently identified and reported to be associated with prostate cancer and chronic fatigue syndrome. To clarify the public health impact of this new virus, it is necessary to develop reliable, specific, and sensitive assays for its presence in blood products.

Methods: We developed the XMRV single copy and single genome sequencing assays (X-SCA and X-SGS, respectively) to detect and quantitate infection and distinguish it from possible background contamination with mouse DNA or RNA. A blinded panel of XMRV-spiked patient samples was used to assess the performance of X-SCA methods for detecting XMRV DNA in whole blood and RNA in plasma. Whole blood was spiked with 3-fold dilutions of XMRV-infected Cw22Rv1 cells to final concentrations of 9900 to 0.5 cells/mL. Plasma was spiked with 5-fold dilutions of XMRV-infected Cw22Rv1 culture supernatants to final concentrations of 2.5x105 to 0.1 virus copies/mL. Six XMRV–control samples were included in both the whole blood and plasma panels. Single genome sequences were obtained from 0.03 mouse cell equivalents and used to develop phylogenetic methods of distinguishing XMRV from endogenous mouse murine leukemia virus (MLV).

Results: All negative control blood and plasma samples were correctly identified as XMRV– by X-SCA. XMRV DNA was detected in 0.5 mL of whole blood spiked with each dilution of infected cells from 9900 to 0.5 cells/mL. Samples were assayed in triplicate and XMRV DNA was detected as expected by Poisson distribution. Sensitivity of X-SCA for detecting XMRV RNA in plasma was >3.3 copies/mL for the limited panel sample volume tested (0.3 mL). All dilutions containing >3.3 XMRV copies/mL were positive by X-SCA. Single genome sequences obtained from mouse cell DNA were closely related to XMRV but could be differentiated phylogenetically by analyzing large fragments of gag or env.

Conclusions: Testing of a blinded panel of XMRV-spiked and control patient samples showed that the single copy DNA and RNA detection assays performed with 100% specificity and high sensitivity. The single sequence assay distinguished between XMRV and closely related MLV in mouse cell DNA. These methods will be applied to evaluate the prevalence of XMRV in relevant cohorts.
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Title: Discordant XMRV Test Results and Non-reproducible Mouse Endogenous Retroviral Detection in an XMRV Prevalence Study
Authors and Affiliations: Timothy Henrich1,2, J Li1,2, D Felsenstein2,3, R Plenge1,2, N Lin2,3, D Kuritzkes1,2, and A Tsibris2,3 1Brigham and Women`s Hospital, Boston, MA, US; 2Harvard Med School, Boston, MA, US; and 3Massachusetts General Hospital, Boston, US

Background: We previously used nested gag PCR amplification strategies to report the prevalence of xenotropic murine leukemia virus-related virus (XMRV) among 293 participants with chronic fatigue syndrome (CFS), HIV, rheumatoid arthritis (RA), organ transplantation, or a general cohort of patients presenting for medical care. XMRV DNA was not detected in any participants’ peripheral blood mononuclear cell (PBMC) samples. Mouse endogenous retrovirus (MERV) sequence was detected in one RA subject but was not reproducibly amplified on subsequent attempts. We have since enrolled 2 CFS participants with outside laboratory evidence of XMRV or gammaretrovirus infection. We used a combined env and gag amplification strategy to identify XMRV or other gammaretroviral DNA in these 2 participants and performed a clonal analysis of the previously reported MERV+ sample.

Methods: CFS subjects fulfilled the Centers for Disease Control and Prevention case definition criteria. One participant tested positive for XMRV and a second participant reported a positive blood test for a gammaretrovirus; both tests were performed at the same outside laboratory. In our laboratory, DNA was extracted from 5 million fresh PBMC; validated primer sets were used in 1 nested env and 2 separate nested gag amplification strategies. A range of input DNA quantities (200 to 600 ng) was used with positive and negative XMRV controls for each amplification. Human beta-globin was amplified to verify DNA integrity. A standard clonal analysis of the prior amplified MERV gag sequence was performed; 6 clones were bi-directionally sequenced.

Results: We could reliably detect 1 env copy and 10 gag copies of XMRV control plasmid. We did not detect XMRV or other gammaretroviruses from 2 CFS participants at any input DNA concentration for both gag and env PCR amplifications. The 6 MERV clones isolated from 1 participant had 90 to 91% sequence identity to recently described CFS-associated murine leukemia virus (MLV)-like sequences. A Bayesian inference of phylogeny demonstrated clustering of MERV sequences separate from MLV-like sequences and the original CFS-associated XMRV sequences.

Conclusions: XMRV was not detected in 2 patients with previous positive test results. Sequence analysis suggested significant differences between a non-reproducibly amplified MERV sequence and CFS-associated MLV-like and XMRV sequences. A standardized, cross-validated amplification strategy for gammaretroviral detection in clinical samples is urgently needed.
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Title: Serologic and PCR Testing of Persons with Chronic Fatigue Syndrome in the US Shows No Association with XMRV
Authors and Affiliations: William Switzer1, H Jia1, H Zheng1, S Tang1, R Garcia2, and B Satterfield2 1CDC, Atlanta, GA, US and 2Cooperative Diagnostics, Greenwood, SC, US

Background: In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus-related virus (XMRV), was reported in 67% of persons from the United States with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. However, 5 subsequent studies in the US, Europe, and China found no evidence of XMRV or murine leukemia virus (MLV) in CFS samples from different patient populations. More recently, another group detected MLV sequences but not XMRV in 87% of US CFS patients. Thus, more work is necessary to investigate the prevalence of these murine retroviruses in persons with CFS.

Methods: We tested blood specimens from 45 CFS cases and 42 persons without CFS from more than 20 states in the US. Plasma specimens were tested for antibodies using a new Western blot assay that utilizes purified XMRV as antigen. Whole blood DNA samples were tested using 2 generic XMRV/MLV PCR tests in the polymerase region and one for gag sequences. Plasma from the CFS patients was also tested for viral RNA using a real-time PCR test that generically detects XMRV/MLV protease sequences. CFS patients were diagnosed using the 1994 research case definition and all had a minimum of 6 months of post-exertional malaise and a high degree of disability.

Results: We found no evidence of either XMRV or MLV in all 45 CFS cases and in the 42 persons without CFS using a comprehensive testing strategy.

Conclusions: Our results, combined with previous negative studies, do not suggest an association of XMRV/MLV in the majority of CFS cases across the US. Differences in patient populations may explain the discordant test results observed in each study. Further work is required to determine the prevalence of XMRV and MLV in persons with CFS and to evaluate a causal link of these viruses found in a subset of CFS cases.
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Title: Extensive Genetic Recombination in the XMRV Genome
Authors and Affiliations: William Switzer1, W Heneine1, M Prosperi2, and M Salemi2 1CDC, Atlanta, GA, US and 2University of Florida, Gainesville, US

Background: Xenotropic murine leukemia virus-related virus (XMRV) is a newly described human retrovirus identified in some persons with prostate cancer, chronic fatigue syndrome (CFS), and in blood donors. More recently a polytropic murine leukemia virus (MLV) has also been found in persons with CFS. It is currently unclear if XMRV or MLV is an etiologic agent of these diseases or simply an incidental infection. To gain insight into the genetic history and evolution of XMRV we performed complex phylogenetic and genetic recombination analyses.

Methods: Prototypic XMRV and MLV complete genomes, including 3 representatives from each MLV host receptor tropism class (ecotropic, polytropic, and xenotropic), were aligned using MAFFT and edited with MEGA. Recombination was inferred using multiple methods including SimPlot, Recombination Detection Program (RDP), split decomposition, and the sliding window PHI test. Substitution saturation was assessed using transition/transversion versus divergence plots, the Xia test for saturation, and likelihood mapping. Phylogenetic relationships were inferred using MEGA and PhyML.

Results: We found significant evidence of recombination in the XMRV genome but each method gave different numbers and locations of recombination breakpoints. Little or no substitution saturation and low phylogenetic noise was seen in the alignment suggesting these artifacts did not influence our results. The sliding PHI test revealed specific regions with highly incompatible phylogenetic reconstructions that were due to recombination and not homoplasy. Bootscan analysis in SimPlot inferred that XMRV is a recombinant of xenotropic, polytropic, and ecotropic viruses across the genome. For example, the 5' and 3' ends of the XMRV p15 gag gene are composed of ecotroptic and xenotropic MLV sequences, respectively. Since this region is commonly targeted for diagnostic PCR tests, phylogenetic analysis of p15 gag sequences may incorrectly infer classification of viral tropism.

Conclusions: Our results suggest that XMRV is a complex mosaic resulting from multiple recombination events, possibly occurring over a significant period of time. More research will be necessary to further investigate the mosaic structure of XMRV and to determine if recombination occurred before or after crossing into humans. In addition, caution should be taken when using small genomic regions for phylogenetic inference of MLV and XMRV tropism since this analysis may be influenced by viral recombination.
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Title: Disease-associated XMRV Sequences Explained by Laboratory Contamination Authors and Affiliations: Stéphane Hué1, E Gray1, A Gall2, A Katzourakis3, CP Tan1, C Houldcroft2, J Garson1, O Pybus3, P Kellam2, and G Towers1 1Univ Coll London, UK; 2Wellcome Trust Sanger Inst, Cambridge, UK; and 3Univ of Oxford, UK

Background: The presence of the retrovirus xenotropic murine leukemia virus-related virus (XMRV) has been reported in prostate cancer (PC) and chronic fatigue syndrome (CFS) patients. Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls.

Methods: To understand the origin of XMRV we screened DNA from mice and human cell lines with Taqman PCR primers described as XMRV-specific. We then sequenced the XMRV-related amplicons from these sources and performed extensive evolutionary analyses including published XMRV and related murine leukemia virus (MLV) sequences.

Results: All 12 mouse strains tested were positive using apparently XMRV-specific Taqman PCR primers targeting a 24-nucleotide deletion in the gag-leader. Nine of 411 human cell lines (2.2%) were positive for xenotropic MLV (MLV-X) and 5 of these were also positive using XMRV gag-leader primers. Phylogenetic analysis of the amplified sequences confirmed that the contaminating viruses are MLV-X distinct from XMRV. Phylogenetic analyses also revealed that pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants between XMRV found in the 22Rv1 PC cell line and Moloney murine leukemia virus (MMLV), a virus that cannot replicate in humans but is commonly encoded within plasmids. Bayesian phylogenies also clearly showed that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with sequences amplified from 22Rv1 cells (posterior probability >0.99). Indeed the cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p <0.001 for pol and env, respectively). These observations are incompatible with the hypothesis that patient XMRV sequences are derived from a process of infectious transmission.

Conclusions: We demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify high-copy sequences in mice. Our analyses provide several independent lines of evidence that XMRV sequences PCR-amplified from PC and CFS patients are the result of PCR contamination from virus found in 22Rv1 cells or encoded in plasmids.
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Title: Identification of a Novel Endogenous Murine Leukemia Virus as an XMRV
Ancestor Authors and Affiliations: Oya Cingoz and John Coffin Tufts University School of Med, Boston, MA, US

Background: XMRV represents the first instance of a gamma-retrovirus detected in human tissues. The sequence similarity between XMRV and murine leukemia viruses (MLV) strongly suggests that XMRV originated from an MLV, commonly found as endogenous proviruses in the genomes of wild and inbred mice. We sought to identify the ancestral element(s) that gave rise to XMRV.

Methods: We used 4 approaches to search for XMRV-like sequences among the ~100 endogenous MLV in mouse genomes: PCR-based assay, single genome amplification, genomic hybridization blots, and in silico searches of sequenced mouse genomes. Ligation-mediated PCR combined with restriction enzyme digestion was used to selectively amplify and sequence a provirus with the gag leader deletion characteristic of XMRV, along with flanking DNA. Using this sequence information, we analyzed the distribution of this potential XMRV ancestor in wild and inbred mice.

Results: We used a PCR assay specific for XMRV sequences, we found no proviruses identical to XMRV among DNAs from 32 inbred strains and 46 wild mice of 16 species. We did, however, find at least one provirus with the XMRV-specific gag leader deletion in the genomes of various mouse strains. This provirus was detected in 17 inbred mouse strains, as well as in 2 wild strains. The strain distribution is in perfect agreement with our previous results using XMRV-specific primers, implying that it is the only provirus in these mice with the characteristic deletion. Sequencing of the full-length provirus revealed intact open reading frames, including a Pmv-related env gene. A ~3.5 kb stretch including gag, pro and part of pol is 100% identical at every base to the XMRV consensus sequence, while the rest of the provirus differs by 10.8%. Interestingly, those regions of XMRV that are different than the provirus are exactly covered by a different endogenous, suggesting that XMRV is a recombinant between these 2 mouse endogenous retroviruses.

Conclusions: Our results indicate that XMRV is not found as a single endogenous MLV, but we report the identification of a Pmv-related MLV provirus that is likely one of the two ancestors that gave rise to it. Although not previously characterized, this provirus is present in many inbred and a few wild mouse species.

PRESENTATION DURING THE SESSION: “VIRUS-CELL INTERACTION & CO-FACTORS”

Title: XMRV Probably Originated through Recombination between 2 Endogenous Murine Retroviruses during in vivo Passage of a Human Prostate Cancer Xenograft
Authors and Affiliations: T Paprotka1, K Delviks-Frankenberry1, O Cingoz2, A Martinez3, H-J Kung3, C Tepper3, W-S Hu1, J Coffin2, and Vinay Pathak*1 1NCI-Frederick, MD, US; 2Tufts Univ Sch of Med, Boston, MA, US; and 3Univ of California, Davis, Sacramento, US

Background: Xenotropic murine leukemia virus–related virus (XMRV) has recently been associated with human prostate cancer (PC) and chronic fatigue syndrome (CFS). However, other studies have failed to detect XMRV in PC and CFS patients. A human PC cell line, 22Rv1, produces XMRV that is virtually identical to virus isolated from PC and CFS patients. The 22Rv1 and CWR-R1 cell lines were derived from a human PC xenograft, CWR22, which was serially passaged in nude mice. To evaluate the genetic variation and evolutionary potential of XMRV, nucleic acid extracts of early and later passages of the CWR22 xenografts and CWR-R1 were analyzed.

Methods: DNA isolated from early (3rd and 7th) and later passage CWR22 xenografts consisted of a mixture of tumor DNA and nude mouse DNA. Short tandem repeat analysis was used to confirm that the tumor DNA were derived from the same person as the 22Rv1 and CWR-R1 cell lines. XMRV-specific PCR primers and qPCR assays were developed and used for the analysis of xenograft and nude mouse nucleic acids. We explored the origin of XMRV by analyzing xenograft-associated nude mouse DNA and DNA from other nude mouse strains; the data revealed the presence of two previously undescribed endogenous proviruses, PreXMRV-1 and PreXMRV-2, which contained >3.2-kb stretches of their genomes with ~99.92% identity to XMRV.

Results: PCR assays showed that both cell lines and later passage xenografts contained XMRV but the early passage xenografts did not, indicating that XMRV was not present in either the original CWR22 tumor or associated nude mouse tissue, but became prevalent in later passage xenografts. Retroviral recombination between PreXMRV-1 and PreXMRV-2 involving a few template switching events can generate a replication-competent virus that differs from XMRV by only 5 nucleotides (99.94% identity). Analysis of 15 nude mouse strains indicated that none contained XMRV, but some strains potentially used to passage the xenograft contained both PreXMRV-1 and PreXMRV-2.

Conclusions: We conclude that XMRV was not present in the original CWR22 prostate tumor but was generated by recombination between PreXMRV-1 and PreXMRV-2 during in vivo passages of the CWR22 xenograft. The probability of an identical recombinant arising multiple times is vanishingly small, raising the possibility that contamination of human samples with XMRV originating from the 22Rv1 cell line is responsible for its reported association with PC and CFS.

POSTERS

Title: Determination of Host Range and Cellular Tropism of XMRV
Authors and Affiliations: Krishnakumar Devadas, MK Haleyur Giri Setty, R Viswanath, O Wood, S Tang, J Zhao, A Dastyar, X Wang, S Lee, and I Hewlett Center for Business and Economic Research, FDA, Rockville, MD, US

Background: Xenotropic murine leukemia virus-related virus (XMRV) is a newly identified retrovirus identified in familial cases of prostate cancer tissue using a virus gene array. Although initial reports have identified XMRV predominantly in the prostate, recent reports of detection of XMRV in blood cells of patients with chronic fatigue syndrome suggests that blood cells could act as a primary target and reservoir for XMRV and help in disseminating infection throughout the body. The aim of this study is to elucidate possible routes of transmission and to determine the host range and cellular tropism of XMRV, particularly in cells derived from the hematopoietic system.

Methods: To determine the host range and tropism of XMRV, culture supernatants containing infectious virus from 22RV-1 or DU145-clone-7 cells were used to infect human cell lines Jurkat, H9, HL60, U937, DU145, LNCaP, and primary monocytes and monocyte-derived macrophages (MDM). Infected cells were monitored for XMRV replication over a period of 5 days. XMRV replication was quantitated by RT-PCR, DNA PCR, and Western blotting. To determine the tropism of XMRV, GHOST(3) cells expressing CD4 and other co-receptors like CXCR4, CCR5, and BONZO, were infected with XMRV and viral replication quantitated by real-time PCR.

Results: Replication of XMRV could be observed in the prostate cancer cell lines DU145 and LNCaP, T cell lines Jurkat and H9, B cell line HL60, and in primary monocytes and MDM. The levels of XMRV transcripts were lower in primary monocytes compared to T cell lines suggesting less efficient replication in these cells. GHOST(3) cells expressing CD4 could support viral replication. However, more viral replication was detected in GHOST(3) cells expressing other co-receptors together with CD4.

Conclusions: Viral replication could be identified in primary hematopoietic cells and cell lines investigated. In addition, viral replication was considerably lower in primary monocytes, suggesting less efficient replication in these cells. Studies with GHOST(3) cells indicate that CD4 and other co-receptors may act in a synergistic manner to enhance XMRV infection. These observations will help to further our understanding of XMRV pathogenesis and provide insights into the modes of transmission involved in XMRV infection.
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Title: Lack of the Detection of XMRV or Polytropic MLV-related Sequences in Blood Cells from HIV-1-infected Patients in Spain
Authors and Affiliations: Joanna Luczkowiak, O Sierra, L Martínez-Prats, S Fiorante, R Rubio, F Pulido, and R Delgado Hosp Univ 12 de Octubre, Madrid, Spain

Background: 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 (CFS). These studies have been controversial since a number of laboratories have been unable to find evidence of XMRV in similar groups of patients. Since XMRV infection has been shown in up to 4% of healthy blood donors, we decided to explore the presence of this agent in a group of patients infected with HIV-1 with a high proportion of intravenous drug use (IDU) and co-infection by hepatitis C virus (HCV).

Methods: We have studied a group of 40 HIV-1- infected patients under follow up in our institution: Of these, 57.5% were men, 52.2% active or past IDU, and 60% were co-infected with HCV (90.4% of IDU were HCV+). DNA was extracted from 400 µL EDTA-whole blood from the group of patients and 10 healthy blood donors. XMRV and polytropic MLV-related virus screening was performed by nested PCR reaction on 500 ng of DNA using 2 sets of primers targeting gag (1st PCR: 419F/1154R, 2nd PCR: MLV-GAG-I-F/MLV-GAG-I-R and MLV-NP116/MLV-NP117) and 1 set of primers targeting env (1st PCR: 5922F/6273R, 2nd PCR: 4672F/7590R). The full-length molecular viral clone of XMRV Vp62 was used as a positive along with primers for human beta-globin (hBG). As a control of sensitivity the region of HIV-1 gp120 corresponding to the V3 loop was amplified by nested PCR in all samples.

Results: The sensitivity of the nested PCR strategy estimated by amplification of XMRV Vp62 was less than 10 copies. All samples from HIV-1-infected patients were positive for V3 amplification. No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls.

Conclusions: XMRV or polytropic MLV-related virus do not appear to be associated with HIV-1 infection in a group of patients with a high proportion of IDU and HCV co-infection. Although we cannot rule out a very low prevalence (95%CI 0 to 10) the role of these viruses as widespread blood-borne agents seems unlikely.
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Title: A Sensitive Real-time PCR Assay for the Detection and Quantification of XMRV
Authors and Affiliations: Laura Li, M Raines, and T Robins Quest Diagnostics Clinical Trials, Valencia, CA, US

Background: Xenotropic murine leukemia virus-related virus (XMRV) was first identified in prostate tissue from prostate cancer patients. One study reported detection of XMRV in 67% of patients with chronic fatigue syndrome (CFS), as compared with 3.7% of healthy controls. Subsequently, several studies failed to detect XMRV in CFS patients, sparking controversy in the field. Therefore, a more sensitive and specific method is needed to resolve the issue. To this end, we have developed a sensitive real-time PCR assay that reliably detects XMRV from CFS patients.

Methods: XMRV RNA was extracted from CFS patients’ peripheral blood mononuclear cells (PBMC) or PBMC/LNCap cells using Trizol reagent. RNA was reverse transcribed using Superscript VILO cDNA synthesis kit. Taqman primers and probe were designed and used in the quantitative real-time PCR assay.

Results: The performance of our quantitative real-time PCR assay was determined, and then it was compared with the current XMRV detection method. Current XMRV detection methods use a nested PCR assay followed by agarose gel detection of the PCR product. These methods are labor intensive and time consuming. A sensitive real-time PCR assay was developed to detect and quantify XMRV within 2 hours. This assay could detect as few as 1 to 2 copies of XMRV RNA molecule in the reaction. In addition, the linear range of the assay was established and quantification of XMRV virus was performed to show that the assay can be used to detect and quantify XMRV RNA simultaneously. Furthermore, specificity of the assay was confirmed by the fact that it detects XMRV but not HIV, hepatitis B virus, or hepatitis C virus. Direct comparison of 1 current method with the quantitative real-time PCR assay was carried out. PBMC from CFS patients were either co-cultured with LNCap cells or not. Viral RNA was extracted and tested with both methods. Both methods could detect XMRV in the co-cultured PBMC/LNCap. However, only the quantitative real-time PCR assay could detect XMRV in non-co-cultured PBMC.

Conclusions: Based on preliminary performance data, this real-time PCR assay could be a sensitive and quantitative tool for detecting XMRV.
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Title: No Evidence for XMRV Infection in Transplant Recipients from Germany
Authors and Affiliations: Klaus Korn and A Knoell University Hospital Erlangen, Germany

Background: Xenotropic murine leukemia virus-related virus (XMRV) is a gamma-retrovirus that was first described in 2006 in prostate cancer tissue. More recently, a strong association of XMRV with the chronic fatigue syndrome (CFS) has been described. In CFS patients, viral DNA has been detected in peripheral blood mononuclear cells (PBMC), suggesting the possibility of blood-borne transmission of XMRV. This has recently led to recommendations to exclude persons with CFS as blood donors in several countries. Therefore, we chose to investigate patients after solid organ transplantation (SOT) or hematopoietic stem cell transplantation (HSCT) for evidence of XMRV infection. These patients may be at an increased risk of XMRV infection due to frequent transfusions of cellular blood products and because of therapeutic immunosuppression, which might reduce their ability to ward off the infection.

Methods: EDTA blood samples from patients after HSCT or SOT that had been submitted for CMV monitoring were selected for XMRV testing. DNA isolation was done using the MagNaPure DNA Large Volume kit. A real-time PCR assay that is able to detect less than 5 copies of XMRV DNA per reaction was used. To monitor extraction efficiency and to control for PCR inhibition, a nonhuman herpesvirus was used as an internal control in all samples. Furthermore, cellular DNA was quantified by real-time PCR targeting the human albumin gene.

Results: Five hundred and three EDTA blood samples from HSCT or SOT recipients were tested for XMRV DNA. Except for 1 sample with an equivocal result (weak signal with threshold cycle of 37.2 in the real-time PCR assay), all samples were negative for XMRV DNA. Unfortunately, no further material was left from the equivocal sample for further analysis. Two other EDTA blood samples obtained from the same patient (a 67-year-old stem cell transplant recipient) 1 week before and 2 weeks after the equivocal samples were negative for XMRV DNA. Therefore, no further investigation by other PCR assays and/or sequence analysis was possible until now.

Conclusions: This study on transplant recipients is in accordance with a number of other studies from several European countries that were unable to find evidence of XMRV infection in patients with prostate cancer and in CFS patients. Although the disease entities are presumably similar in Europe and the US, there is a striking difference in the number of reported cases of XMRV infection. It would be interesting to know if such a difference also exists between transplant recipients from these geographic areas.
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Title: Limited Evidence of XMRV Infection in Different Populations in Spain, Including Patients with CFS and PC
Authors and Affiliations: Miguel Arredondo1, J Hackett2, F deBethencourt3, A Treviño1, D Escudero4, A Collado5, P Labarga1, P Swanson2, V Soriano1, and C de Mendoza1 1Hosp Carlos III, Madrid, Spain; 2Abbott Labs, Abbott Park, IL, US; 3Hosp La Paz, Madrid, Spain; 4Hosp Germans Trias i Pujol, Barcelona, Spain; and 5Hosp Clin, Barcelona, Spain

Background: The recent discovery of xenotropic murine leukemia virus-related virus (XMRV) and its association with chronic fatigue syndrome (CFS) and prostate cancer (PC) has generated enormous interest. However, conflicting results have been obtained by different investigators. In this study, we examined populations of archived specimens from Spain for evidence of XMRV infection using serological and molecular methods.

Methods: Plasma and peripheral blood mononuclear cells (PBMC) were collected from 1103 patients: 437 CFS and/or fibromyalgia (FM), 69 PC, 149 HIV, 31 human T cell lymphotropic virus type 1 (HTLV-1), 81 chronic hepatitis B, 72 chronic hepatitis C, 18 autoimmune disease, and 246 blood donors. Plasma samples were evaluated for the antibodies to XMRV transmembrane protein using a prototype ARCHITECT XMRV p15E research assay (Abbott Diagnostics, Abbott Park, IL). DNA extracted from PBMC were tested for the presence of XMRV gag sequences using nested PCR following the Lombardi protocol. Samples that were gag PCR positive were further evaluated using nested PCR for XMRV env sequence.

Results: Of the 1103 plasma samples, 3 (0.3%) were p15E seroreactive with a sample/cutoff (S/CO) of >1.0: 2 HTLV-1 patients (S/CO 1.2 and 4.8) and 1 chronic hepatitis C patient (S/CO 1.04). Of the 662 PBMC DNA tested by PCR, 4/69 (5.8%) samples from PC patients with stage T1 (2), T2 (1), or T3 (1) cancer were repeatedly positive for XMRV gag sequences. However, XMRV env sequences could not be PCR amplified from DNA of these 4 patients. Moreover, no patients had both p15E antibodies and detectable XMRV gag sequences.

Conclusions: In this study, limited evidence of XMRV infection was identified in a large population of Spanish patients with various medical conditions including CFS and PC previously associated with XMRV infection. However, further confirmatory studies are required. Screening for the presence of gp70 antibodies using a more sensitive serological assay is in progress.