Human Parvovirus PARV4 in Blood and Plasma Products Jacqueline Fryer National Institute for Biological Standards and Control SoGAT XX

Identification of PARV4 and detection in pooled plasma Originally identified in a homeless drug abuser with acute viral infection syndrome; co-infected with HBV (Jones et al, J Virol. 2005) PARV4 and related genotype (PARV5) detected in 4-5% of recent manufacturing plasma pools; higher prevalence in older pools Both viruses found in approximately equal proportions The highest viral loads in pools are ~ 106 copies/ml plasma No correlation between B19V and PARV4/5 viral loads PARV4 and PARV5 are highly conserved (92% nucleotide identity), but share limited homology with B19V and HBoV (Fryer et al. EID 2006; Fryer et al. Transfusion 2007)

Phylogenetic analysis of parvovirus genomes Primate, chipmunk, bovine parvoviruses Adeno-associated and avian parvoviruses Human bocavirus, canine and bovine parvoviruses Parvoviruses from carnivores, rodents and pig Porcine parvovirus 2 Genus Erythrovirus Genus Dependovirus Genus Bocavirus Genus Amdovirus Genus Parvovirus

Are PARV4 & PARV5 present in products derived from these plasma pools? Clotting factor VIII concentrates 175 lots, 12 products, 10 manufacturers Expiry dates 1974-2005 (plasma sourced ~5 yrs prior to expiry date) Reconstituted in sterile H20, 1ml extracted on MagNA Pure LC Extractor PARV4/5 detection by conventional PCR (ORF2 primers) Viral loads determined by consensus real-time PCR

PARV4 and PARV5 in factor VIII concentrates Product / Manufacturer Expiry date No. of lots tested Purification process Virus inactivation No. of positive lots by PCR PARV4/PARV5 B19V 1/A 1974-1978 37 Precipitation None 3 23 2/B 1976-1977 2 1 3/C 1976-1978 5 4/D 1977-1978 5/E 1977-1980 55 14 9 6/C 1985 Dry heat (68 °C, 72 h) 4/F Precipitation and adsorption Wet heat (heptane) (60 °C, 20 h) 7/E 1985-1987 8 8/A 1986 4 Precipitation (plus further purification) Steam treatment (60 °C, 10 h) 9/EGH 1997-2004 16 Monoclonal antibody Pasteurisation (60 °C, 10 h) 10/I 1998-2002 13 Solvent/detergent 7 11/I 1999-2003 Dry heat (80 °C, 72 h) 12/J 2001-2005 18 Affinity chromatography Solvent/detergent, dry heat (80 °C, 72 h) No. positive/total tested 28/175 70/175 Percent positive 16% 40%

Prevalence of PARV4, PARV5 and B19V in factor VIIIs manufactured over the past 30-35 years PARV4 and PARV5 B19V PARV4/PARV5 and B19V Negative for these viruses

Analysis of PARV4/5-positive factor VIII concentrates Prevalence of PARV4/5 vs. age of product Pre-1990 expiry: 23% PARV4/5 (45% B19V) Post-1990 expiry: 2% PARV4/5 (30% B19V) Viral loads PARV4/5; up to 3 x105 copies/ml product B19V; up to 2.5 x108 copies/ml product Sequence analysis 7/28 PARV4 19/28 PARV5 2/28 both PARV4 and PARV5

Summary PARV4 and PARV5 present in coagulation factor VIII products manufactured over past 30-35 years Prevalence of PARV4 and PARV5 higher in ‘older’ products (expiring pre-1990) compared to recent products (expiring post- 1990) (23% vs. 2%) Increased blood safety in early 1980s (HIV epidemic); Screening / exclusion of ‘high risk’ donors PARV4 sequences from 30-35 year period highly conserved (>99% nucleotide identity), same for PARV5 Prevalence of PARV5 higher in ‘older’ factor VIII products, analogous to parvovirus B19 genotypes 1 & 2 Implications for recipients of plasma and plasma products in terms of PARV4/5 contamination are unknown Nothing yet known of role of PARV4/5 in human disease

Acknowledgements NIBSC Sally Baylis Tony Hubbard Nita Shah Morag Ferguson Janice Blinder Philip Minor Blood Systems Research Institute, California/ University of California Eric Delwart