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


METHODS
A new B19-specific Light Cycler (LC) reverse transcription-polymerase 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 degrees 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.


CONCLUSIONS
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.
Prikhod'ko
Grigori G
GG
Plasma Derivatives Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA. PrikhodkoG@usa.redcross.org
Vasilyeva
Irina
I
Reyes
Herbert
H
Wong
Susan
S
Brown
Kevin E
KE
Jameson
Thomas
T
Busby
Thomas F
TF
D001681:Biological Assay; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D016731:Erythema Infectiosum / Q000382:microbiology / Q000517:prevention & control / Q000635:transmission; D005069:Evaluation Studies as Topic; D006358:Hot Temperature; D006801:Humans; D010322:Parvoviridae Infections / Q000097:blood / Q000821:virology*; D016732:Parvovirus B19, Human / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D038301:Virus Inactivation
D004279:DNA, Viral