1. SOGAT 25-05-2005 Presence (absence) of genotype 2 and 3 Erythrovirus DNA in manufacturing plasma Theo Cuypers, Marco Koppelman, Margret Sjerps, Henk Reesink Sanquin Plasma Products and Diagnostic Services

2. SOGAT 25-05-2005 Genotypes detection Erythroviruses by Parvo B19 DNA test (1) Oct 2001 notification by EP (group 6B) for change Monograph Anti-D Immunoglobulin; NAT for Parvo B19 DNA, effective per January 2003. Fractionation facilities anticipated on introduction in a relative short period (had to deal with plasma stocks). No guidance on detection of other Erythroviruses than Parvo B19 virus. Jan. 2003 official notification of Monograph Human Anti-D Immunoglobulin per January 2004.

3. SOGAT 25-05-2005 Genotypes detection Erythroviruses by Parvo B19 DNA test (2) June 2003. Validation Guideline Parvo B19 NAT for Official Medicine Control laboratories (OMCL’s): test including detection of Erythrovirus sequences genotype 2 and 3 July 2003. EDQM. Testing for Erythrovirus sequences is added as ‘important notice for information’ and not ‘mandatory’ April 2004. Proficiency testing study (PTS) on B19 Virus DNA NAT of plasma pools Recommendation to OMCL’s: improve NAT methods to cover also B19 variant sequences (A6, V9)

4. SOGAT 25-05-2005 Erythrovirus variants V9 (genotype 3) Nguyen et al.1999. Lancet 352: 1524. Heegaard et al. 2001 J. Med. Virol. 65: 362- 367. A6 (genotype 2) Nguyen et al. 2002. Virology 301: 374 Belonging to the human Erythrovirus genus, genotype 1 (Parvo B19 virus), genotype 2 (prototype A6) en genotype 3 (prototype V9).

5. SOGAT 25-05-2005 Phylogenetic relationships

6. SOGAT 25-05-2005 Specificity of test kits for Erythrovirus genotype 2 and 3 quantification Parvovirus B19 quantification kit Roche Reliable quantification of genotype 1. Genotype 2 and 3 are not detected 1,2,3. RealArt Parvo B19 LC PCR Artus. Genotype 1 and 2 reliable quantified3. Genotype 3 under-quantified 1000x1. Lower limit of detection: 5000 kop/reaction 2. In house tests PTS study EDQM 3. In house protocols detected genotype 1; genotype 2 missed by 50 % of the protocols. Genotype 3 was not included. 1 Baylis et al. J. of Virol. Methods 2004 121, 7 2 Hokynar et al J. of Clin. Microbiol. 2004 42, 2013 3 PTS052 “B19 Virus DNA Testing of Plasma Pools by NAT” September 2004

7. SOGAT 25-05-2005 NAT testing for Erythroviruses genotype 2 and 3 sequences in plasma pools Plasma pools ~ 100.000 Danish blood donations, no genotype 3 sequences 1 Plasma pools ~ 140.160 Finnish blood donations, no genotype 2 (and genotype 3) sequences 2 52 plasma pools from 9 different manufacturers, no genotype 2 and 3 sequences detected 3 Plasma pools ~950.000 Dutch blood donations, no genotype 2 and 3 sequences 4 1 Heegaard et al. J. Med. Virol. 2001, 65 362-367 2 Hokynar et al. J. of Clin. Microbiol. 2004, 42 2013-2019 3 Baylis et al. J. of Virol. Methods 2004 121, 7-16 4 Koppelman et al. 2004 manuscript in preparation

8. SOGAT 25-05-2005 Additional NAT testing manufacturing pool for genotype 2 and 3. Sanquin Plasma-products Specific NAT test for Erythrovirus genotype 2 and 3. Test with no cross reactivity with Parvo B19 DNA (genotype 1). Retested 321 manufacturing pools Sept 2003 Oct 2004 (~950.000 donations) No genotype 2 and 3 sequences detected.

9. SOGAT 25-05-2005 Erythrovirus genotype 2 and 3 in coagulation factor concentrate 1. Haemophilia Centre Bonn Germany 1. 13 commercially available plasma-derived coagulation factor concentrates (181 lots). Period collected 2000-2003. Analyzed with specific NAT tests for genotype 1 and 2 in final products. Genotype 1 DNA 77/181 (42.5 %) and genotype 2 DNA 2/181 (1.1 %). Genotype 2 always co-contaminated with genotype 1. 1 Schneider et al. 2004 Thromb Haemost 92 838-845

10. SOGAT 25-05-2005 Conclusions (1) Data on testing of plasma pools for Erythrovirus genotype 2 and 3 sequences do not indicate that the specificity of applied Parvo B19 tests should be extended to these new molecular variants. Additional data on the prevalence and load of Erythroviruses genotype 2 and 3 in plasma pools and individual donations should be collected in a research setting. Additional studies are dependent on publicly available reference materials for these molecular variants (WHO, EDQM, and commercial suppliers).

11. SOGAT 25-05-2005 Conclusions (2) The new variant viruses are not classified by the International Committee on Taxonomy of Viruses. Terminology in literature is confusing: Family Parvoviridae, genus Erythrovirus, genotype 1 (Parvo B19), genotype 2 (A6 like), genotype 3 (V9 like) or Parvovirus B19 gt 1, gt 2, and gt 3.