1. Identification of the New Human Retrovirus XMRV

2. Disclosures Research Support –Abbott Laboratories Patent Licenses and Consulting –Abbott Laboratories

3. The Hereditary Prostate Cancer 1 Gene Encodes the Antiviral Protein RNase L Carpten, J.. et al., Nature Genetics, 30, 181-4, 2002

4. Prostate Cancer Tissue RNA/DNA Virochip Hybridization Pattern & RT- PCR Urisman, et al. PLoS Pathogens 2006 Red VP= homozygous for R462Q RNase L Variant Retrovirus Discovery in Prostate Cancer

5. Related to Murine Leukemia Virus Silverman et al., NATURE REVIEWS Urology, 2010

6. Urisman et al., PLoS Pathogen, 2006; Dong et al., PNAS, 2007 XMRV Xenotropic Murine Leukemia Virus-Related Virus

7. Green: XMRV Blue: DAPI Red: Cytokeratin Scale Bar=10  m XMRV in Prostate Cancer Tissue Urisman A. PLoS Pathog. 2006 Mar;2(3):e25 Stromal fibroblast Dividing stromal cell Stromal hematopoetic cell

8. Semen-Derived Factor (SEVI) Enhances Infectivity Hong, Klein et al, J Virol 83:6995, 2009 XMRV in human prostatic secretions semen enhances infections

9. Effect of Androgen on XMRV Androgen Stimulates XMRV ReplicationCasodex Suppresses XMRV Replication ARE in XMRV Promoter Dong & Silverman, J Virol 84:1648, 2010

11. PCR and sequencing novel XMRV isolates XMRV proteins detected in activated PBMC, T cells and B cells (immunoblots) Activated PBMC from CFS patients, but not from controls, transmitted XMRV to LNCaP cells in co- culture experiments Plasma from CFS patients, but not from controls, transmitted XMRV to LNCaP cells Circulating antibody against XMRV envelope protein was detected in CFS patients, but not in control individuals XMRV in CFS Cases [Lombardi et al., Science (2009)]

12. XMRV in Chronic Fatigue Syndrome XMRV detected by nPCR in 67% of patients vs. 3.7% of controls (n = 101 and 218) Lombardi et al, Science 326:585, 2009 Electron Micrograph of C-type Retrovirus Particles Transmitted from CFS activated T cells to LNCaP

13. Association of XMRV with prostate cancer and other diseases XMRV RNase L MethodsCell Types or fluid Prostate Cancer R462Q Urisman et al. (2006) YesYesVirochip,RT-PCR, FISH, IHCStroma Dong et al. (2007)YesYesIntegration site mappingn.d. Kim et al. (2008) YesYesIntegration site mappingn.d. Fischer et al. (2008) Yes (low)NoRT-PCRn.d. Sfanos et al. (2008) Non.d.PCRn.d. Knouf et al. (2009) YesYesRT-PCRepithelial (22Rv1) Hong et al. (2009) YesNoqRT-PCR, nested RT-PCR Prostate secretions Schalberg et al. (2009) YesNoqPCR, IHCEpithelial Hohn et al. (2009) NoNoPCR, qRT-PCR, ELISAn.d. Arnold et al. (2010) YesYesab, IHC, FISHStromal fibroblasts D’Arcy (2008) NoNoRT-PCRn.d. Furuta (2009) Yesn.d.Immune response, RT-PCRPBMC SUMMARY:9/12 5/10 Chronic fatigue syndrome Lombardi et al. YesNoPCR, WB, culturing virus, abPBMC, T-, B-cells, plasma Erlwein et al. Non.d.PCRwhole blood Groom et al.Non.d.PCR, abPBMC, sera Van Kuppeveld et al.Non.d.PCRPBMC Switzer et al.Non.d.PCR, WB, ELISAPBMC, sera SUMMARY:1/5 0/1 Amyotrophic lateral sclerosis McCormick et al. NoNoRT-PCRPBMC, CSF

14. Large Differences in Detection Rates (possible reasons) Lab contaminationLab contamination Geographical distributionGeographical distribution Sequence variantsSequence variants Clinical criteria for patient selectionClinical criteria for patient selection Lack of standardized methodsLack of standardized methods Lack of widely available positive control human specimensLack of widely available positive control human specimens

15. Animal Protocol: IV Inoculation of Rhesus Macaques with XMRV

16. XMRV Infection in Primates Early TargetsLate Targets Lymphoid Organs Peripheral Blood (CD4+) Spleen & Lymph Nodes Bone Marrow Large & Small Intestine Lymphoid Organs --- Spleen & Lymph Nodes --- Large & Small Intestine Lung (macrophages) Pancreas (epithelium)--- Genital Tract Prostate (epithelium) Testis Seminal vesicle Genital Tract Prostate (stroma only) Testis Seminal vesicle & Epididymis Cervix & Vagina (epithelium)

17. Possible Role of XMRV in Prostate Cancer and CFS Silverman et al., NATURE REVIEWS Urology, 2010

18. Conclusions XMRV is a novel retrovirus associated with prostate cancer and CFS in humans in some, but not all, studies –All individuals at risk regardless of RNaseL/HPC1 genotype XMRV establishes both acute and chronic infections in primates –Prostate epithelium is an early target; stroma late –CD4 + and other blood cell types infected XMRV growth is fueled by androgen _ Potential oncogenic mechanism XMRV might be transmitted by blood transfusion –CFS pts deferred from donation in Canada, Australia, and NZ Causal link to any human disease remains to be established

19. Acknowledgements Cleveland Clinic –Eric Klein –Jaydip Das Gupta –Beihua Dong –Ross Molinaro (Emory) Abbott Diagnostics -John Hackett Jr -Gerald Schochetman -Xiaoxing Qui -Sushil Devare Yerkes/Emory –Francois Villinger –Prachi Sharma TGEN -John Carpten, Jeff Trent JHMI -William Isaacs USC - Graham Casey Whittemore Peterson Institute - Judy Mikovits, Vincent Lombardi NCI- Francis Ruscetti, Sandra Ruscetti University of Ulm - Jan Munch, Frank Kirchhoff UCSF –Joseph DeRisi –Don Ganem –Anatoly Urisman –Nicole Fischer