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  • 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
  • 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
  • Discordant XMRV Prevalence in Persons with CFS
    • 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)
    • Zero Prevalence:
    Erlweinet al.2010- 0/186 CFS from UK
    - proviral nested PCR; gag and pol)
    Groom et al. 2010 - 0/170 CFS and 0/395 controls from UK
    - DNA PCR (gag andenv)
    - 1/565 showed neutralizing activity (CFS patient), but was nonspecific
    van Kuppeveldet 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
  • Methods
    • Developed WB assay for antibody detection using polytropicMuLV-infected
    (PMLV) and uninfected HeLa cells
    - same assay format used successfully to identify human infection with
    simian foamy virus
    - plasma tested at 1:50 dilution
    • Developed highly sensitive and specific nested PCR assays in multiple viral
    genes (gag, pol)
    - samples screened with nested gag and polPCR tests using 1 ug DNA
    input (integrity confirmed by B-actin PCR)
    • Developed sensitive mouse sequence specific qPCR to detect contamination
    with mouse DNA
    - XMRV positive DNA samples tested for mouse contamination
  • Strong Reactivity of MuLV antiserum to WB Antigen, CDC
    100
    80
    60
    50
    40
    30
    20
    α GaLV (p30; 1:50)
    α MuLV whoe virus
    α Ra MuLV Env
    Pre-immune
    Pre-immune
    Pre-immune
    32,000
    64,000
    32,000
    64,000
    16,000
    16,000
    32,000
    64,000
    16,000
    4000
    2000
    1000
    8000
    2000
    8000
    1000
    4000
    250
    500
    4000
    2000
    8000
    1000
    250
    500
    250
    500
    gp69/71(Env)
    pr65(Gag)
    gp69/71
    HeLa/XMLV
    pr65
    p30(CA)
    p15E(TM)
    p30
    p15(MA)
    HeLa
    α Friend MuLV (whole virus)
    α Rauscher MuLV (gp69/71)
    α XMRV (whole virus)
    Rat α SSFV Env (32,000), not shown
  • High Sensitivity and Specificity of PCR Assays, CDC
    Test
    Gene Sensitivity2 Specificity Notes
    XGAGN110 copies (34/34, 100%)0/41 US BD (100%)Urismannet al.
    XPOLN10 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
    1. N, nested PCR
    2. Sensitivity determined using XMRV VP62 plasmid diluted in 1 ug human DNA or VP62 RNA
  • Murine
    PCR
    XMRV nPCR3
    Serology
    Sample Type Total XGAG XPOL MCOX2 WB
    Prostate DNA 162 1(0.6) 3(1.9)0/3 (0) -
    Plasma 162 - - - 0/162 (0)
    1. Percentages in parentheses
    2. Dashes indicate test not performed on these sample types
    3. nPCR, nested PCR
    Rare XMRV Infection in Prostate Cancer, CDC
    Switzer, et al. CROI, 2010
  • Study Population
    • 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
    • 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:
    - 18 DNA
    - 19 plasma
    - included three persons (17%) with sudden onset
  • Lab Testing
    • Blood specimens tested using a combination of molecular and serologic
    assays
    • Blinded testing and included positive and negative controls
    at independent labs
    • 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:
    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
    recombinant XMRV Env and Gag proteins
    • Nested gag PCR at Blood Systems Research Institute (BSRI), San Francisco, CA
    - 3 copies/250 ng DNA assay sensitivity
  • Absence of XMRV in CFS and Healthy Persons from the US
  • 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
  • Absence of XMRV antibodies in CFS patients
    by Western blot analysis, CDC
    α Ra MuLV (1:500)
    α Fr MuLV (1:500)
    Pre-immune
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
    200
    CFS
    100/120
    80
    gp69/71(Env)
    Infected
    HeLa
    pr68(Gag)
    60
    50
    40
    p30(CA)
    30
    20
    200
    Uninfected
    HeLa
    100/120
    80
    60
    50
    40
    30
    20
  • Absence of XMRV antibodies in CFS patients and healthy persons by ELISA using XMRV rec-proteins, RKI
    0.8
    OD healthy
    0.7
    OD positive control
    OD CFS
    0.6
    Assay cut-off
    0.5
    OD 492/620
    0.4
    0.3
    0.2
    0.1
    0
    W3
    G11
    G1
    G7
    G3
    G5
    G8
    G9
    G2
    G4
    G6
    W19
    W34
    W4
    W6
    W9
    W7
    W1
    W11
    W26
    Mouse
    sera
    G10
    G12
    G14
    G13
    G15
    G16
    W23
    W17
    W32
    W16
    W18
    W20
    W31
    W33
    W35
    W37
    W10
    W15
    W21
    W22
    W25
    W30
    W36
    W13
    W14
    W28
    W12
    W27
    W29
    0.8
    0.7
    0.6
    0.5
    OD 492/620
    0.4
    0.3
    0.2
    0.1
    0
    G39
    G70
    G37
    G31
    G32
    G34
    G35
    G38
    G66
    G67
    G71
    G73
    G74
    G19
    G20
    G23
    G24
    G27
    G28
    G29
    G30
    G33
    G36
    G40
    G42
    G44
    G45
    G48
    G49
    G58
    G59
    G62
    G63
    G65
    G68
    G69
    G72
    G75
    G17
    G21
    G22
    G25
    G26
    G41
    G46
    G47
    G50
    G60
    G61
    G64
    Mouse
    sera
  • Absence of XMRV polymerase sequences in
    CFS patients, CDC
    XMRV 103 copies
    XMRV 10 copies
    1° PCR
    Neg DNA
    H2O
    H2O
    CFS
    2° PCR
    ß-actin
    CFS
    H2O
    10-1 to 10-4
    BD DNA
  • Absence of XMRV gag sequences in CFS patients, BSRI
    XMRV 10 copies
    XMRV 3 copies
    XMRV 1 copies
    Neg DNA
    CFS
    H2O
    2° PCR
    GAPDH
  • Absence of XMRV DNA in Additional Populations
    Kunstmanet al. 2010, AIDS
    0/996 men from the Chicago MACS (562 HIV+, 434 HIV-)
    proviralqPCR; gag (Singh primers)
    Gaoet al. 2010, ICEID– Gen-Probe and ARC
    0/1435 blood donors from ARC, NC
    0/44 HIV-1+ blood donors
    rtTMA; DNA and RNA
  • Conclusions and Summary
    • 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
    of CFS patients and matched healthy controls
    • 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
    possibility of false negative results caused by divergent viruses
    • 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
    of CFS cases
    • 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
    of XMRV in CFS, blood donors, and the general population.
  • Acknowledgements
    Bill Switzer, Hongwei Jia, Shoahua Tang, Hao Zheng, Anupama Shankar, Bill Reeves, Rebecca Falkenberg, Walid Heneine
    Graham Simmons
    Oliver Hohn and Norbert Bannert
    Robert Silverman
    Sandy Ruscetti
    Ila Singh
    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.