1. Update on CDC XMRV Activities R. Michael Hendry, D.Sc. Chief, Laboratory Branch DHAP, NCHHSTP, CDC Blood Products Advisory Committee July 26, 2010 The findings and conclusions in this presentation are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention

2. CDC Activities: Retrovirology, CFS, and Blood Safety Retrovirology: Laboratory Branch Division of HIV/AIDS Prevention National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention CFS, Epidemiology: Chronic Viral Diseases Branch Division of High-Consequence Pathogens and Pathology National Center for Emerging and Zoonotic Infectious Diseases Blood and Tissue Safety: Office of Blood, Organ, and other Tissue Safety Division of Healthcare Quality Promotion National Center for Emerging and Zoonotic Infectious Diseases

3. High prevalence: High prevalence: Lombardi et al. 2009 - 68/101 (67%) CFS; 5/218 (3.6%) healthy persons (US) - RNA PCR from plasma, proviral PCR, Flow-based antibody testing, culture (activated PBMCs, plasma) - RNA PCR from plasma, proviral PCR, Flow-based antibody testing, culture (activated PBMCs, plasma) Zero Prevalence: Zero Prevalence: Erlwein et al. 2010 0/186 CFS from UK Erlwein et al. 2010 - 0/186 CFS from UK - proviral nested PCR; gag and pol) - proviral nested PCR; gag and pol) Groom et al. 2010 - 0/170 CFS and 0/395 controls from UK - DNA PCR (gag and env) - DNA PCR (gag and env) - 1/565 showed neutralizing activity (CFS patient), but was nonspecific - 1/565 showed neutralizing activity (CFS patient), but was nonspecific van Kuppeveld et al. 2010 - 0/32 CFS and 0/43 controls from the Netherlands - DNA RT-PCR (int) and nested PCR (gag) - cDNA step first to increase assay sensitivity Discordant XMRV Prevalence in Persons with CFS

4. Methods Developed WB assay for antibody detection using polytropic MuLV-infected Developed WB assay for antibody detection using polytropic MuLV-infected (PMLV) and uninfected HeLa cells - same assay format used successfully to identify human infection with simian foamy virus simian foamy virus - plasma tested at 1:50 dilution Developed highly sensitive and specific nested PCR assays in multiple viral Developed highly sensitive and specific nested PCR assays in multiple viral genes (gag, pol) - samples screened with nested gag and pol PCR tests using 1 ug DNA input (integrity confirmed by B-actin PCR) input (integrity confirmed by B-actin PCR) Developed sensitive mouse sequence specific qPCR to detect contamination Developed sensitive mouse sequence specific qPCR to detect contamination with mouse DNA - XMRV positive DNA samples tested for mouse contamination

5. Strong Reactivity of MuLV antiserum to WB Antigen, CDCHeLa/XMLV HeLa10080 60 50 40 30 20 α Friend MuLV (whole virus) 250500 1000 4000 20008000 16,000 32,000 α GaLV (p30; 1:50) 64,000 Pre-immune α Rauscher MuLV (gp69/71) 250500 1000 40002000 8000 16,000 32,00064,000 Pre-immune 250500 1000 400020008000 16,000 32,000 α MuLV whoe virus 64,000Pre-immune α Ra MuLV Env α XMRV (whole virus) p30 gp69/71 pr65 p30(CA) p15E(TM) p15(MA) gp69/71(Env) pr65(Gag) Rat α SSFV Env (32,000), not shown

6. High Sensitivity and Specificity of PCR Assays, CDC GeneSensitivity 2 SpecificityNotes XGAG N 1 10 copies (34/34, 100%)0/41 US BD (100%)Urismann et al. XPOL N 10 copies (32/34, 94.1%)0/41 US BD (100%)generic MCOX210 copies (12/12, 100%) 0/117 US BD (100%) 1 cell equivalent 5 copies (12/12, 90%) Proviral Mouse-specific Test 1. N, nested PCR 2. Sensitivity determined using XMRV VP62 plasmid diluted in 1 ug human DNA or VP62 RNA

7. Rare XMRV Infection in Prostate Cancer, CDC 1. Percentages in parentheses 2. Dashes indicate test not performed on these sample types 3. nPCR, nested PCR Sample TypeTotalXGAGXPOLMCOX2WB Prostate DNA1621(0.6)3(1.9)0/3 (0)- Plasma162---0/162 (0) XMRV nPCR 3 MurinePCRSerology Switzer, et al. CROI, 2010

8. Study Population Archived, anonymous plasma and matching PBMCs/DNA from 51 persons with Archived, anonymous plasma and matching PBMCs/DNA from 51 persons with CFS and 56 matched healthy controlsavailable for testing CFS defined using 1994 International Research Case Definition CFS defined using 1994 International Research Case Definition Population based (telephone interviews and clinical evaluation): Population based (telephone interviews and clinical evaluation): - 11 CFS and 26 healthy controls from Wichita, KS - 22 CFS and 30 healthy controls from Georgia (rural, urban, metro) - 3/33 CFS (9%) reported sudden onset Physician referred CFS persons from Bibb County, GA with clinical evaluation: Physician referred CFS persons from Bibb County, GA with clinical evaluation: - 18 DNA - 19 plasma - included three persons (17%) with sudden onset

9. Lab Testing Blood specimens tested using a combination of molecular and serologic Blood specimens tested using a combination of molecular and serologicassays Blinded testing and included positive and negative controls Blinded testing and included positive and negative controls at independent labs WB at CDC using a polytropic MuLV as antigen and comparison of reactivity WB at CDC using a polytropic MuLV as antigen and comparison of reactivity to uninfected antigen to determine viral-specific seroreactivity Nested PCR to detect two gene regions at CDC: Nested PCR to detect two gene regions at CDC: gag = XMRV specific but can detect polytropic MuLV polymerase (pol) = generic for xenotropic and polytropic MulV - 10 copies/ 1 ug DNA sensitivity of each assay XMRV EIA and IFA at Robert Koch Institute (RKI), Berlin, Germany using XMRV EIA and IFA at Robert Koch Institute (RKI), Berlin, Germany using recombinant XMRV Env and Gag proteins Nested gag PCR at Blood Systems Research Institute (BSRI), San Francisco, CA Nested gag PCR at Blood Systems Research Institute (BSRI), San Francisco, CA - 3 copies/250 ng DNA assay sensitivity

10. Absence of XMRV in CFS and Healthy Persons from the US SpecimensCDC pol PCR CDC gag PCR CDC WBRKI EIARKI IFABSRI gag PCR CFS0/50 0/511/510/10/50 Healthy Controls0/56 0/531/530/10/56

11. Absence of XMRV Antibodies in Additional Populations Tested at CDC 0/13 HTLV-1/2 + 0/7 HIV-1 + 0/6 HIV-1/HIV-2 dual + 0/121 US Blood Donors 0/20 US IVDU 0/20 “positive” plasmas from WPI

12. Pre-immune α Ra MuLV (1:500) α Fr MuLV (1:500) 123456789101112 100/120 80 60 50 40 30 200 20 100/120 80 60 50 40 30 200 20 p30(CA) gp69/71(Env) pr68(Gag) InfectedHeLa UninfectedHeLa Absence of XMRV antibodies in CFS patients by Western blot analysis, CDC CFS

13. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.70.8G17G19G20G21G22G23G24G25G26G27G28G29G30G31G32G33G34G35G36 G37 G38 G39 G40G41G42G44G45G46G47G48G49G50G58G59G60G61G62G63G64G65G66G67G68G69 G70 G71G72G73G74G75 Mouse sera sera W1 W3 W4W6W7W9 W10 W11 W12W13W14 W15W16 W17 W18 W19 W20W21W22 W23 W25 W26 W27W28W29 W30W31 W32 W33 W34 W35W36W37 G1 G2G3G4G5G6 G7G8G9 G10 G11 G12G13G14G15G16 Mouse 0 0.1 0.2 0.3 0.4 0.6 0.7 0.8 0.5 OD CFS Assay cut-off OD healthy OD positive control OD 492/620 Absence of XMRV antibodies in CFS patients and healthy persons by ELISA using XMRV rec-proteins, RKI

14. Absence of XMRV polymerase sequences in CFS patients, CDC 1° PCR 2° PCR ß-actin XMRV 10 copies XMRV 10 3 copies Neg DNA H2OH2OH2OH2O H2OH2OH2OH2O CFS 10 -1 to 10 -4 BD DNA H2OH2OH2OH2O CFS

15. Absence of XMRV gag sequences in CFS patients, BSRI GAPDH 2° PCR XMRV 10 copies XMRV 3 copies Neg DNA H2OH2OH2OH2O XMRV 1 copies CFS

16. Absence of XMRV DNA in Additional Populations Kunstman et al. 2010, AIDS Kunstman et al. 2010, AIDS 0/996 men from the Chicago MACS (562 HIV+, 434 HIV-) 0/996 men from the Chicago MACS (562 HIV+, 434 HIV-) proviral qPCR; gag (Singh primers) proviral qPCR; gag (Singh primers) Gao et al. 2010, ICEID Gen-Probe and ARC Gao et al. 2010, ICEID – Gen-Probe and ARC 0/1435 blood donors from ARC, NC 0/1435 blood donors from ARC, NC 0/44 HIV-1+ blood donors 0/44 HIV-1+ blood donors rtTMA; DNA and RNA rtTMA; DNA and RNA

17. Conclusions and Summary Developed highly sensitive assays for detection of human infection with Developed highly sensitive assays for detection of human infection with XMRV and other MuLVs We did not find any evidence of infection with XMRV in our study population We did not find any evidence of infection with XMRV in our study population of CFS patients and matched healthy controls PCR and serologic methods performed independently in three laboratories PCR and serologic methods performed independently in three laboratories blinded to the clinical status of the study participants Testing included generic PCR and two serology assays which reduces Testing included generic PCR and two serology assays which reduces possibility of false negative results caused by divergent viruses Differences in patient population, complexities of CFS, lab methods used, etc. Differences in patient population, complexities of CFS, lab methods used, etc. may explain the contrasting results of our study and those of Lombardi et al. However, our results do not support an association of XMRV with the majority However, our results do not support an association of XMRV with the majority of CFS cases More research is needed to determine the prevalence of XMRV in the general More research is needed to determine the prevalence of XMRV in the general population, to investigate its transmissibility, and to standardize testing across labs More studies are needed to better understand the prevalence and significance More studies are needed to better understand the prevalence and significance of XMRV in CFS, blood donors, and the general population.

18. Bill Switzer, Hongwei Jia, Shoahua Tang, Hao Zheng, Anupama Shankar, Bill Reeves, Rebecca Falkenberg, Walid Heneine Acknowledgements The findings and conclusions in this presentation are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Robert Silverman Sandy Ruscetti Ila Singh Oliver Hohn and Norbert Bannert Graham Simmons