1. Detection of Parvovirus B19 Variants in Factor VIII Concentrates Mei-ying W. Yu 1, Yansheng Geng 1, Susan Wong 2, Kevin Brown 3 ; 1 CBER/FDA, U.S.A.; 2 NHLBI/NIH, U.S.A.; 3 HPA, UK SoGAT XIX Bern, Switzerland 15 June 2006

2. Procedures for Detecting Genotypes 2 and 3 Use a pair of consensus primers located in NS1 region to detect genotypes 1- 3 Discriminate between genotype 1 and genotypes 2/3 by using MfeI restriction enzyme digestion of the PCR product –Genotype 1 of B19, cleaved (36 bp + 67 bp fragments) –Genotypes 2 and 3, not cleaved (103 bp) Identify whether it is genotype 2 or 3 by cloning (786-bp PCR product containing C-terminal NS1 region and the VP1u region), sequencing, and phylogenetic analysis [Servant et al, J Virol 2002; 76: 9124-34 and Nguyen et al, Virology 2002; 301: 374-80]

3. MfeI Restriction Enzyme Digestion of PCR products

4. Detection of Parvovirus B19 Genotype 2 in a Wet-Heated, Intermediate-Purity Antihemophilic Factor (Human) (AHF) Made in 1997 # AHF Lots Viral # PCR (+) # MFeI Tested Inactivation not cleaved 60 (3 products made 27 (45%+) 0 before 1984) No 53 (6 products made before 1998) Yes 34 (64%+) 1* 89 (6 products made during 2002-2004) Yes 18 (20%+) 0 * By sequencing and phylogenetic analysis, one intermediate-purity AHF lot subjected to wet-heating was found genotype 2 positive, 10 3 geq/mL. The lot was not co-contaminated with genotype 1.

5. Phylogenetic Analysis of Sequences of VP1u Region AY345134 New3 New2 New1 AJ249431 A6-AY064475 IM81-AY9034347 LALI-AY044266 V9-AX003421 AJ249430 AY582125 AJ249421 AHF1 CBER1 PAT1 AY386330 AY504945 CBER3 WHO3 WHO2 AF162273 NC00083 Au-M13178 CBER2 WHO1 0.05 Genotype 2 Genotype 1

6. Detection of PARV5 in a Wet-Heated, Intermediate-Purity AHF Made in 1988 The same AHF lots representing 6 AHF products were tested for the presence of PARV4/5 using nested primers derived from VP2 region. Only one lot of an intermediate-purity AHF subjected to wet-heating made in 1988 was PARV4/5-positive. PARV5 sequence was identified by both Southern blot hybridization and sequencing. The lot also contained low-level of genotype 1 DNA.

7. Summary Detection of B19 Related Variants in AHF # Lots Tested Viral Inactivation Variant Type 60 (3 products made No No lot positive for Genotype 2, 3, or before 1984)PARV 4/5 53 (6 products made 1 lot positive for Genotype 2 before 1998) Yes 1 lot positive for PARV5 89* (6 products made during 2002 - 2004) Yes No lot positive for Genotypes 2, 3, or PARV4/5 * Lots were also tested for the presence of Bocavirus. However, no lot was positive.

8. Conclusion One intermediate-purity AHF subjected to wet-heating made in 1997 was positive for genotype 2, 10 3 geq/mL. It was not co-contaminated with B19 genotype 1. One lot of the same type of product made in 1988 was positive for PARV5. The lot was co-contaminated with a low level of genotype 1. In 89 lots representing 6 AHF products made in 2002 - 2004, no B19 related variants (which include bocavirus) were found. The prevalence of B19 variants was very low. Thus, the impact of these variants on viral safety of plasma-derived product remains to be determined.