title: Quantitation of HTLV-I and II proviral load using real-time quantitative PCR with SYBR Green chemistry.
abstract: BACKGROUND
  Human T-lymphotropic virus type I (HTLV-I) is linked etiologically with adult T cell leukemia/lymphoma and HTLV-I-associated myelopathy/tropical spastic paraparsis (HAM/TSP). Human T-lymphotropic virus type II (HTLV-II) is associated with HAM/TSP and, in HIV coinfected patients only, rare cases of cutaneous T cell lymphoma. Proviral load may be important in the pathogenesis of HTLV-associated disease.
  
  
  METHODS
  A real time quantitative PCR assay using SYBR Green intercalation was established. Primers targeting the tax region were standardized against MT2 and MOT cell line DNA for HTLV-I and HTLV-II, respectively. HTLV-I/II copy number was normalized to the amount of cellular DNA by quantitation of the HLA-DQ alpha gene. We measured proviral load in peripheral blood mononuclear cells (PBMCs) in a large cohort of 120 HTLV-I and 335 HTLV-II seropositive former blood donors. We also assessed the intra- and inter-assay reproducibility of the assay.
  
  
  RESULTS
  Proviral load for HTLV-I infected patients ranged from 3.1 x 10(0) to 1.8 x 10(5)copies/10(6) PBMCs with a mean of 1.6 x 10(4) and a median of 3.0 x 10(3). HTLV-I was undetectable in 7 of 120 cases (5.8%). Proviral load for HTLV-II infected patients ranged from 1.1 x 10(0) to 1.0 x 10(6)copies/10(6) PBMCs with a mean of 2.8 x 10(4) and a median of 5.0 x 10(2). HTLV-II was undetectable in 31 out of 335 cases (9.3%).
  
  
  CONCLUSIONS
  The assay has excellent dynamic range from 10(6) to 10(0)copies/reaction, good intra- and inter-assay reproducibility, and a lower limit of detection of a single copy per reaction. The sensitivity and high dynamic range allow determination of a broad range of HTLV-I/II proviral load in clinical subjects. This assay will facilitate the study of the relationship between proviral load and pathogenesis.
authors.affiliation: Blood Systems Research Institute, San Francisco, CA 94118, USA.
authors.forename: Tzong-Hae
authors.initials: TH
authors.lastname: Lee
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Chafets
authors.forename: Michael P
authors.initials: MP
authors.lastname: Busch
authors.forename: Edward L
authors.initials: EL
authors.lastname: Murphy
mesh_terms: D052160:Benzothiazoles; D004279:DNA, Viral / Q000032:analysis; D003959:Diamines; D015490:HTLV-I Infections / Q000276:immunology / Q000821:virology*; D015491:HTLV-II Infections / Q000276:immunology / Q000821:virology*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000502:physiology*; D015367:Human T-lymphotropic virus 2 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000502:physiology; D011804:Quinolines; D019562:Viral Load
chemical_list: D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I