1. Perrine Caillet-Fauquet Laboratoire de Virologie Moléculaire Université Libre de Bruxelles B19 production in hepatocarcinoma cells and neutralization

2. B19 BIOLOGY n Predilection for infecting dividing cells n Low genetic complexity of B19 links its replication highly to cell factors (S phase and differentiation) n Strong tropism for haematopoietic cells such as marrow or fetal liver erythroïd progenitor cell. Endocytosis via the P blood group antigen (B19 receptor) and a co-receptor (  integrin) R&D

3. Primary cultures n Bone marrow cells (human-monkey) n CFU-e peripheral blood n Umbilical cord blood cells n Fetal liver cells Cell lines n Blast cell linesUT-7 TF-1 M-07 B1647 n Megacaryotic leukemia cell line MB-02 n Megacaryotic JK-1 n Erythroid cell line KU812 n Erythroid cell line KU812 Ep6 n Erythroid cell line KU812F in hypoxia (6%O 2 ) n Hepatocarcinoma cell lines R&D

4. INCUBATION 2 hours CENTRIFUGATION Elimination of the remaining input and 3X wash addition of medium INCUBATION 3 days CENTRIFUGATION Supernatant: DNA extraction Nested-PCR B19 Virus (WHO 99/800, NISBC ) + EPO KU812F cells Infected cells 6%O 2 or 20%O 2 R&D KU812F pluripotent cells

5. 0 1 2 3 0110100100010000 Virus input (IU) B19 detectable end-point (log dilution) B19 production in KU812F cells (+epo) Hypoxia compared to Normoxia 6%O 2 20%O 2 R&D

6. HepG2 is Human hepatoblastoma cell line HepG2(or HuH7) Cellular model for B19 production Erythrovirus B19 B19 2 hours 37°C Cells Washing 3x Centrifugation 24, 48, 72 hours 37°C Supernatant PCR POSITIVE DNA Extraction PCR Amplification Cells R&D WHO 99/800, NISBC

7. B19 production in HepG2 cells and HuH7 0 1 2 3 4 5 6 7 244872 Post infection time (h) Detectable end-point (log dilution) 0.1 IU10 IU100 IU0 IU A HepG2 0 1 2 3 4 5 6 7 244872 Post infection time (h) Detectable end-point (log dilution) 0.1 IU10 IU100 IU0 IU A HuH7 R&D

8. Measure of apotosis: % annexin V positive hepatocarcinoma cells R&D

9. 183bp RT-PCR:spliced mRNA VP Actin B19 transcription in HepG2 100 0 NS1 VP1 VP2 P6 VP1 Map units Protein: 11 kDa B19L2:381-404 (Brunstein et al 2000) B19L21:2082-2067 B19L20:2066-2050 Unspliced 1702 Splice donor: 406 splice acceptors : 1910 183bp amplicon sizes 72h48h24h B19: input IU 10 2 11 1 Hep NI Hep NI 72h48h24h R&D

10. 0 1 2 3 4 5 CONTROL+ ANTI-P Detectable end-point (log dilution) HepG2 HuH7 C Inhibition of B19 infection in HepG2 by antibodies anti- P blood group antigen Cells + Anti-P 1 hour 4°C B19 Cells 2 hour 4°C Washing 3x 48 hours 37°C Supernatant DNA Extraction Nested-PCR ? R&D

11. B19 48 hours at 37°C HepG2 B19 Neutralization ANTIBODIES + 16 hours at RT Culture Supernatant DNA extraction NESTED PCR Washing 3x ? HepG2 R&D

12. RABBIT MODEL OF ANTI-B19 NEUTRALIZING ANTIBODIES Method : l Selection of potential VP/NS epitopes l Rabbit immunization l Purification of rabbit IgG l Elisa on peptides and Commercial Kit (Biotrin) l Testing in the cellular model R&D

13. B19 NEUTRALIZATION by ANTIBODIES IN IVIG Concentration of IVIG to obtain 50% virus neutralization - 10 ng/ml for MULTIGAM - 300 ng/ml for SANDOGLOBULIN Method: B19 DNA (10 3 IU) from a single plasma donation incubation Over Night at room temperature with IVIG concentrations : 3x10 -4 to 300 µg/ml R&D

14. Conclusions 1.New assay for parvovirus B19 infectivity and neutralization. 2.Use of B19 as relevant model for virus inactivation methods. R&D

15. DCF Red Cross M. Di Giambattista V. Hougardy R. Laub Université Libre de Bruxelles M.-L. Draps Y. de Launoit Aknowlegments R&D