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  • First let me briefly review some basic background information for HTLV
    Like HIV, following cross-species transmission HTLV has spread globally. It is estimated that HTLV now infects about 22 million people worldwide
    HTLV-1 is endemic in West Central Africa, the Caribbean, Brazil, Columbia, Peru, Japan, Melanesia, and Papua New Guinea.
    Collectively, STLV-1 and HTLV-1 are called primate T-lymphotropic viruses type 1 (PTLV-1)
    HTLV-2 and is endemic in Amerindians and thus HTLV-2 is believed to have moved out of Africa, through Asia, and across the Bering Straight with these ancient indigenous populations as they migrated into the Americas.
    HTLV-2 more recently has become endemic in IDUs in the U.S., Europe, and Vietnam during the Vietnam war.
    HTLV-2 is estimated to infect several million people. Collectively, HTLV-2 and STLV-2 are called PTLV-2.
    Two new HTLVs were found recently in primate hunters from Cameroon – now 3 cases of HTLV-3 infection. Collectively, HTLV-3 and STLV-3 are called PTLV-3.
    Today I will be discussing the new HTLV we call HTLV-4. Currently there is not a simian counter part of this virus but if there was collectively they would be called PTLV-4

    Like HIV, HTLV infection is transmitted from person-to-person via sex, from mother-to-child mostly by breast feeding, and by contact with HTLV-infected blood via transfusion and IVDU (needle sharing).
    HTLV is less pathogenic than HIV and is associated with disease in less than 5% of infected persons.
    HTLV-1 is associated with T-cell leukemia/lymphoma which has a long incubation period and usually manifests in adulthood. About 40% of ATLL cases present with some form of cutaneous involvement as shown here.
    HTLV-1 is also associated with a severe neurologic disorder called HTLV-1 associated myelopathy - also known as tropical spastic paraparesis. HAM/TSP can have a more acute course with an incubation period of 3 months to 3 years although some cases can also appear decades later. HAM/TSP is a demyelinating disease of the spinal cord which affects the lower extremities with patients developing problems with walking (as shown here) and they also have bladder and bowel incontinence.
    HTLV-1 has also been associated with a variety of inflammatory and autoimmune diseases most noteably uveitis.
    In contrast, HTLV-2 is less pathogenic than HTLV-1, and has been recently found to be associated with a neurologic disease similar to HAM/TSP.
  • Thanks to the many members of my lab for their hard work over the years and to Tom Folks and Walid Heneine for their continued support.

    Nathan Wolfe and Don Burke and their colleagues for the tremendous amount of work involved in establishing and maintaining the field sites in Cameroon and for providing the primate hunter samples.
    Susan Marriott for helping to characterize the HTLV-3 and HTLV-4 Tax proteins.

    Thanks for your time and attention!
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    1. 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. 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. 3. • 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: Erlwein et 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 and env) - 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. 4. Methods • 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 - 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 pol PCR 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
    5. 5. Strong Reactivity of MuLV antiserum to WB Antigen, CDC HeLa/XMLVHeLa 100 80 60 50 40 30 20 α Friend MuLV (whole virus) 250 500 1000 4000 2000 8000 16,000 32,000 αGaLV(p30;1:50) 64,000 Pre-immune α Rauscher MuLV (gp69/71) 250 500 1000 4000 2000 8000 16,000 32,000 64,000 Pre-immune 250 500 1000 4000 2000 8000 16,000 32,000 αMuLVwhoevirus 64,000 Pre-immune αRaMuLVEnv 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. 6. High Sensitivity and Specificity of PCR Assays, CDC Gene Sensitivity2 Specificity Notes XGAGN 1 10 copies (34/34, 100%) 0/41 US BD (100%) Urismann et al. XPOLN 10 copies (32/34, 94.1%) 0/41 US BD (100%) generic MCOX2 10 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. 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 Type Total XGAG XPOL MCOX2 WB Prostate DNA 162 1(0.6) 3(1.9) 0/3 (0) - Plasma 162 - - - 0/162 (0) XMRV nPCR3 Murine PCR Serology Switzer, et al. CROI, 2010
    8. 8. Study Population • Archived, anonymous plasma and matching PBMCs/DNA from 51 persons with CFS and 56 matched healthy controls available 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
    9. 9. 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
    10. 10. Absence of XMRV in CFS and Healthy Persons from the US Specimens CDC pol PCR CDC gag PCR CDC WB RKI EIA RKI IFA BSRI gag PCR CFS 0/50 0/50 0/51 1/51 0/1 0/50 Healthy Controls 0/56 0/56 0/53 1/53 0/1 0/56
    11. 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. 12. Pre-immune αRaMuLV(1:500) αFrMuLV(1:500) 1 2 3 4 5 6 7 8 9 10 11 12 100/120 80 60 50 40 30 200 20 100/120 80 60 50 40 30 200 20 p30(CA) gp69/71(Env) pr68(Gag) Infected HeLa Uninfected HeLa Absence of XMRV antibodies in CFS patients by Western blot analysis, CDC CFS
    13. 13. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 G17 G19 G20 G21 G22 G23 G24 G25 G26 G27 G28 G29 G30 G31 G32 G33 G34 G35 G36 G37 G38 G39 G40 G41 G42 G44 G45 G46 G47 G48 G49 G50 G58 G59 G60 G61 G62 G63 G64 G65 G66 G67 G68 G69 G70 G71 G72 G73 G74 G75 Mouse sera W1 W3 W4 W6 W7 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22 W23 W25 W26 W27 W28 W29 W30 W31 W32 W33 W34 W35 W36 W37 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 Mouse sera 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 controlOD492/620OD492/620 Absence of XMRV antibodies in CFS patients and healthy persons by ELISA using XMRV rec-proteins, RKI
    14. 14. Absence of XMRV polymerase sequences in CFS patients, CDC 1° PCR 2° PCR ß-actin XMRV10copies XMRV103copies NegDNA H2O H2O CFS 10-1 to 10-4 BD DNA H2O CFS
    15. 15. Absence of XMRV gag sequences in CFS patients, BSRI GAPDH 2° PCR XMRV10copies XMRV3copies NegDNA H2O XMRV1copies CFS
    16. 16. Absence of XMRV DNA in Additional Populations • Kunstman et al. 2010, AIDS • 0/996 men from the Chicago MACS (562 HIV+, 434 HIV-) • proviral qPCR; gag (Singh primers) • Gao et al. 2010, ICEID – Gen-Probe and ARC • 0/1435 blood donors from ARC, NC • 0/44 HIV-1+ blood donors • rtTMA; DNA and RNA
    17. 17. 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
    18. 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