Quantification of viral inactivation by photochemical treatment with amotosalen and UV A light, using a novel polymerase chain reaction inhibition method with preamplification.
BACKGROUND
In evaluating a photochemical treatment process for inactivating parvovirus B19, there lacked simple culture methods to measure infectivity. The recently developed enzyme-linked immunospot (ELISpot) infectivity assay uses late-stage erythropoietic progenitor cells and is labor intensive and time consuming. We evaluated a novel, efficient polymerase chain reaction (PCR) inhibition assay and examined correlations with reductions in infectivity.


METHODS
Contaminated plasma was treated with 150 micromol/L amotosalen and 3 J/cm(2) ultraviolet A light and then tested for DNA modification using conventional PCR inhibition and a novel preamplification approach. The novel assay subjected the samples to preamplification cycles using long-template PCR, followed by quantitative PCR (QPCR) inhibition detection. Both approaches were tested for correlations with reductions in viral infectivity by comparing ELISpot assay results of identical samples.


RESULTS
The B19 preamplification inhibition assay showed detection ranges of 2-2.5 log and demonstrated quantitative correlation with up to a 5.8-log reduction in viral infectivity in ELISpot results. Conventional PCR detected a >5 log reduction in amplification, correlated with a 4.4-log reduction in viral infectivity. A range of 4-log inhibition of hepatitis B virus DNA amplification was also achieved.


CONCLUSIONS
The results demonstrated that a novel preamplification QPCR assay is a useful tool for predicting reductions in infectivity after photochemical treatment. This assay was extended to show utility in circumstances where practical in vitro assays are unavailable for the determination of the efficacy of pathogen inactivation.
Allain
Jean-Pierre
JP
University of Cambridge, Cambridge, United Kingdom.
Hsu
Jocelyn
J
Pranmeth
Manisha
M
Hanson
Deborah
D
Stassinopoulos
Adonis
A
Fischetti
Lucia
L
Corash
Laurence
L
Lin
Lily
L
D004279:DNA, Viral / Q000235:genetics; D011564:Furocoumarins / Q000494:pharmacology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010322:Parvoviridae Infections / Q000097:blood / Q000821:virology; D016732:Parvovirus B19, Human / Q000187:drug effects* / Q000502:physiology / Q000528:radiation effects*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D014466:Ultraviolet Rays; D038301:Virus Inactivation / Q000187:drug effects / Q000528:radiation effects
D004279:DNA, Viral; D011564:Furocoumarins; C118577:amotosalen