Evaluation of a new LightCycler reverse transcriptionpolymerase chain reaction infectivity assay for detection of human parvovirus B19 in dry-heat inactivation studies

Evaluation of a new LightCycler reverse transcriptionpolymerase chain reaction infectivity assay for detection of human parvovirus B19 in dry-heat inactivation studies
June 2005
Grigori G. Prikhod'ko1, Irina Vasilyeva1, Herbert Reyes1, Susan Wong1, Kevin E. Brown1, Thomas Jameson1, and Thomas F. Busby1
Transfusion
Volume 45 Issue 6 Page 1011
Blackwell Synergy
1From the Plasma Derivatives Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland; and the Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
BACKGROUND: Human parvovirus B19 (B19) is a widely distributed infectious agent, which causes a variety of illnesses including erythema infectiosum (fifth disease) especially in children, arthritis, aplastic crisis, and hydrops fetalis. B19 can be transmitted from asymptomatic blood donors to recipients of their blood components. Fifth disease has been reported in patients receiving red blood cells, platelets, solvent/detergent-treated plasma, and clotting factor concentrates.
STUDY DESIGN AND METHODS: A new B19-specific Light Cycler (LC) reverse transcriptionpolymerase chain reaction (RT-PCR) infectivity assay was developed for quantitative analysis of the infectivity of B19 in virus validation studies. The cycling conditions and the primers of the new assay were designed to amplify spliced RNA forms but not precursor RNA or B19 genome. One 50 percent infectious dose, determined on UT7/Epo-S1 cells of low passage, equaled 3.74 ? 0.1 log international units of B19 DNA.
RESULTS: The efficiency of the dry-heat process (100?C) on inactivation of B19 spiked and lyophilized with fibrinogen, a major component of the clotting factor concentrate and hemostatic dressing products, was investigated by use of B19-specific LC RT-PCR infectivity assay. At 1.3 to 1.7 percent residual moisture of fibrinogen, the infectivity of B19 was reduced dramatically by 3.3 to 5.1 log for 1 and 2 hours of dry-heat treatment, respectively. B19 infectivity was reduced 1.5, 2.8, and 3.8 log for 1, 2, and 3 hours of dry-heat treatment, respectively, at 0.5 to 0.7 percent residual moisture level.
CONCLUSION: These findings suggest that level of residual moisture of lyophilized fibrinogen with B19 spike correlated with a different resistance of B19 to dry-heat treatment, and that low moisture may stabilize virus against heat.

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