title: Development of a sensitive PCR inhibition method to demonstrate HBV nucleic acid inactivation.
abstract: BACKGROUND
  The evaluation of pathogen reduction technologies with relevant viruses currently contaminating the blood supply is limited by the availability of high-titer virus inocula and sensitive in vitro or in vivo infectivity assays. Because HBV infectivity can only be assessed by in vivo studies with chimpanzees, a sensitive PCR inhibition assay was developed to measure PEN110 inactivation of HBV.
  
  
  METHODS
  PCR amplification of 1.1 kb of HBV genome was optimized to determine DNA damage introduced by treatment with PEN110 in RBCs. Inactivation of duck HBV (DHBV) in RBCs, with measurement of the in vitro infectivity, was performed to validate the PCR assay.
  
  
  RESULTS
  The PCR was highly specific and sensitive for amplification of the HBV genome and used to demonstrate a reduction of at least 7.2 and 8.1 log geq per mL within the first 18 hours of PEN110 treatment. PEN110 inactivation of DHBV was also achieved within the first 18 hours with a reduction factor of at least 5.0 log tissue culture infectious dose 50 percent per mL, suggesting that PCR inhibition is an alternative to infectivity assays.
  
  
  CONCLUSIONS
  This study establishes PCR inhibition as a reasonable approach to assess the efficiency of PEN110 inactivation of human pathogens with human plasma donations that have been found to contain high titers of relevant agents during different stages of infection.
authors.affiliation: VI Technologies, Inc, Watertown, MA, USA.
authors.forename: Shahika
authors.initials: S
authors.lastname: Aytay
authors.forename: Asa
authors.initials: A
authors.lastname: Ohagen
authors.forename: Michael R
authors.initials: MR
authors.lastname: Busch
authors.forename: Bernadette
authors.initials: B
authors.lastname: Alford
authors.forename: John R
authors.initials: JR
authors.lastname: Chapman
authors.forename: Aris
authors.initials: A
authors.lastname: Lazo
mesh_terms: D000818:Animals; D001782:Blood Donors; D004279:DNA, Viral / Q000097:blood; D004912:Erythrocytes / Q000821:virology; D006509:Hepatitis B / Q000175:diagnosis / Q000517:prevention & control / Q000635:transmission; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D017053:Infection Control / Q000379:methods*; D002679:Pan troglodytes; D011073:Polyamines / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D038301:Virus Inactivation / Q000187:drug effects*
chemical_list: D004279:DNA, Viral; C451719:PEN 110; D011073:Polyamines