1. Testing for Parvovirus B19 - Broadening the Assay to Cover Variants
Sally Baylis, NIBSC
SoGAT XVII

2. Screening Plasma Pools for Parvovirus B19 - an OMCL Perspective
European Pharmacopoeia Monographs:
“Human anti-D immunoglobulin” & “Human anti-D immunoglobulin for intravenous administration” (Jan. 2004)
“Human plasma (pooled and treated for virus inactivation)” (July 2004)
Plasma pools should contain not more than 104 IU/ml parvovirus B19 DNA

3. Variant Erythroviruses
V9 isolated from a child with transient aplastic crisis (Nguyen et al., 1998, 1999)
LaLi, K71 dermal isolates (Hokynar et al., 2002)
A6 isolated from an anaemic HIV-positive patient (Nguyen et al., 2002)
D91.1 isolated from a child with transient aplastic crisis (Servant et al., 2002)
Classification proposed by Servant et al., (2002) based upon sequence analysis of the NS1/VP1 region

4. Genetic Diversity of Erythroviruses: Analysis of the NS1/VP1 Region
Servant et al., J. Virol., 2002

5. Roche Parvovirus B19 Quantification Kit-
Genotype 1
Fluorescence (F2/Back
Fluorescence
(F1/F2)
Genotype 2
NTC
Genotype 3
M NTC M
Cycle Number
Cycle
Number
Region amplified: NS1

6. Artus RealArtTM Parvo B19 LC Kit
Cycle Number
Fluorescence (F2/Back -
F1)
Genotype 3
Genotype 2
Genotype 1
NTC
M NTC M
Region amplified: VP1

7. Sensitivity of Detection of Different Erythrovirus Genotypes

8. In-house Erythrovirus TaqMan Assay
Consensus assay, primers & probe directed to the NS1 gene
Manufacturing plasma pools (Europe, North America)
Extraction using the MagNA Pure (Total Nucleic Acid, large volume) & real-time PCR on the LightCycler
Standard curve – WHO International Standard for parvovirus B19 (99/800)

9. In-house Erythrovirus TaqMan Assay
Genotype 3
Genotype 2
Genotype 1
Fluorescence (F1/F2)
NTC
Cycle Number
NTC
Genotype 1
Genotype 3
Genotype 2
Temperature C
Fluorescence –
d(F1)/ dT

10. Conclusions
The commercial assays have limitations in the detection and quantification of the variant erythroviruses
Of 58 plasma pools screened with the Roche & in-house TaqMan assays, results for parvovirus B19 are in agreement
No evidence currently for the presence of variant erythroviruses in manufacturing pools examined by NIBSC

11. Discussion
Primer & probe design affects ability to detect and quantify variant viruses
Compliance with EP threshold concentration of 104 IU/ml may be compromised
Clinical significance, prevalence & geographical distribution of erythrovirus variants is still largely unknown
What are the implications in detecting a pools with high titres of a variant erythrovirus?

12. Acknowledgements Kevin Brown, NIH
Daniel Candotti & Jean-Pierre Allain, Cambridge
Kati Hokynar & Klaus Hedman, University of Helsinki
Annabelle Servant & Antoine Garbarg-Chenon, Paris