1. Novel and Related Variant Parvoviruses in Human Plasma Jacqueline Fryer and Sally Baylis National Institute for Biological Standards and Control Eric Delwart Blood Systems Research Institute, California/ University of California SoGAT XIX

2. Detection of Human Parvovirus 4 (PARV4) Originally identified in a homeless drug abuser with symptoms of; fatigue, vomiting and diarrhoea, sore throat, neck stiffness and joint pains, also infected with HBV (Jones et al, 2005), using DNase SISPA technique. Originally identified in a homeless drug abuser with symptoms of; fatigue, vomiting and diarrhoea, sore throat, neck stiffness and joint pains, also infected with HBV (Jones et al, 2005), using DNase SISPA technique. Highly specific primers for ORF1 of PARV4 were designed to ensure absence of cross-reactivity with B19 Highly specific primers for ORF1 of PARV4 were designed to ensure absence of cross-reactivity with B19 137 plasma pools from different manufacturers were tested for PARV4 DNA by PCR 137 plasma pools from different manufacturers were tested for PARV4 DNA by PCR

3. Extraction and Detection of PARV4 DNA in Pooled Plasma Automated MagNA Pure LC Extractor (Total Nucleic Acid) 1 ml Plasma Pool PARV4 DNA Detection by Conventional & Real-Time PCR

4. Specificity of ORF1 Primers and Screening Manufacturing Plasma Samples for PARV4

5. Analysis of Plasma Pools for the Presence of PARV4 Sequences by PCR Manufacturer No. positive/no. analysed A5/12 B0/7 C0/9 D2/6 E0/14 F0/21 G0/50 H0/16 I0/2

6. Viral Loads in Plasma Pools Testing Positive for PARV4 Sequences by PCR Positive pool Manufacturer PARV4 viral load (genome copies/ml plasma) Human erythrovirus viral load (IU/ml plasma) 1A 5 x 10 5 Negative 2D<500Negative 3A 3.9 x 10 6 * 140 4A<500*340 5A 2.1 x 10 4 * Negative 6A<500*Negative 7D Not determined * Sequences represent PARV5 and not PARV4 (confirmed by cloning and sequencing of amplicons).

7. Phylogenetic Analysis of a 499 bp Sequence from ORF1 of PARV4 & PARV5 ~92% nucleotide identity between PARV4 and PARV5 ~92% nucleotide identity between PARV4 and PARV5 ~46% nucleotide identity with human parvoviruses B19 and human bocavirus (HBoV) ~46% nucleotide identity with human parvoviruses B19 and human bocavirus (HBoV) PPV2 BPV3 B19 A6 D91.1 V9 RMPV LTMPV PTMPV CHIPMUNKPV PARV4 PARV5

8. Conclusions Novel parvovirus PARV4 and variant PARV5 identified in (5%) plasma pools, used in the manufacture of blood products Novel parvovirus PARV4 and variant PARV5 identified in (5%) plasma pools, used in the manufacture of blood products PARV4 and PARV5 titres not a high as human parvovirus B19, nor as frequent PARV4 and PARV5 titres not a high as human parvovirus B19, nor as frequent PARV4 and PARV5 are highly conserved but share limited homology with human parvovirus B19 and HBoV PARV4 and PARV5 are highly conserved but share limited homology with human parvovirus B19 and HBoV Nothing yet known of the role of PARV4 and PARV5 in human disease Nothing yet known of the role of PARV4 and PARV5 in human disease (Fryer et al. Emerg Infect Dis. 2006 Jan;12(1):151-4)

9. Blood donors from Los Angeles area summer 2005 300 pools of 16 29 positives (9.6% or 0.6% if divided by 16). 200 single donations 4 positives (2%) Subjects with symptoms of acute viral infection or highly exposed (MSM, IDU) 16/195 subjects positive (8%) (including 1 PARV5) Increasing prevalence in symptomatic/exposed subjects may reflect pathogenic viral infection OR higher exposure to blood borne viruses (e.g. like GBV-C) Prevalence of PARV4 virus using nested PCR

10. 10 20 30 40 50 60 70 80 90 100 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ gttgatggccctgtggttagCACCCAGCATCAAGAAGCTTTGCAGACAAGAATAACCATGTTTCAGTTTCAGAGAATGGTTCCGGATGGCTTAGCTCCAC PARV4........y..................................G........................................................ Poolx16 24/29........y........................................................................................... Poolx16 2/29........y..................................R........................................................ Poolx16 1/29........y........................T.........G........................................................ Poolx16 1/29........y..................................G................................................R....... Poolx16 1/29........y..................................G........................................................ Single donation 3/4........y..................................G.....................................T.................. Single donation 1/4........y..................................G........................................................ Symptomatic/high risk 7/16........y........................................................................................... Symptomatic/high risk 3/16........y........................................................................................... Symptomatic/high risk 1/16........y..............................................T............................................ Symptomatic/high risk 1/16........y................R.................G........................................................ Symptomatic/high risk 1/16........y..................................G...R.................................................... Symptomatic/high risk 1/16........y.............................C....G........................................................ Symptomatic/high risk 1/16........y..............T........G......C..................................G........C...........C..T. Symptomatic/high risk 1/16 --......T..............T........G......C..................................G........C...........C..T. PARV5 110 120 130 140 150 160 ---------+---------+---------+---------+---------+---------+- TTCCTGAAGAGGAAGTGAGAAGCTTTTTTAAGCTAGgtgaacaggaactgaatatgaaagg PARV4............................................................. Poolx16 24/29............................................................. Poolx16 2/29............................................................. Poolx16 1/29............................................................. Single donation 3/4............................................................. Single donation 1/4............................................................. Symptomatic/high risk 7/15............................................................. Symptomatic/high risk 3/15...............................A............................. Symptomatic/high risk 1/15............................................................. Symptomatic/high risk 1/15.C....................T...........T.......................... Symptomatic/high risk 1/15.C....................T...........T.......................... PARV5 Genetic variation among PARV4 Non-synonymous polymorphism (Q>R)

11. Acknowledgements NIBSC Nita Shah Nita Shah Morag Ferguson Morag Ferguson Janice Blinder Janice Blinder Philip Minor Philip Minor Blood Systems Research Institute, California/ University of California Eric Delwart Eric Delwart