1. Active B19 virions production in hepatoblastoma and hepatocarcinoma cell lines: amplification and genomic stability. Op de beeck A. 1, Draps M.-L. 1, Baurin S. 2, Timmerman D. 2, Branckaert Th. 2, Caillet-Fauquet P. 1, Laub R. 2 Laboratory of Virology, Medicine Faculty, Free University of Brussels 1. R&D- Central Department for Fractionation, Brussels 2

2. Quantification of B19 Direct qPCR in patient samples Infection model : Quantification of B19 mRNA and/or DNA in infected cells (red blood cell progenitor lineage) Infectivity model : Measure production of infectious B19 new particles in cell culture (HepG2, Huh-7)

3. HepG2 Human hepatoblastoma cell line Huh-7 human hepatocarcinoma cell line HepG2 and Huh-7 cellular models for B19 production Erythrovirus B19 B19 2 hours 37°C Cells Washing 3x 24, 48, 72 hours 37°C Supernatant PCR POSITIVE DNA Extraction PCR Amplification Cells WHO 99/800, NIBSC Caillet-Fauquet et al, Transfusion 2004; 44:1340-3.

4. Detection of B19 in the supernatant of HepG2 and Huh-7 B19 : plasma WHO 99/800 Multiplicity of infection (MOI) : 0.1-100 IU/5 10 5 cells : low M.O.I. ! Minimal infectious dose : 0.1 to 1 IU in HepG2 Detection by Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258) 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

5. Viral progeny is infectious B19 : C39 positive donation devoid of anti-B19 IgG or IgM Input of each run : 100 IU/ 5 10 5 cells (m.o.i. = 0.002) Quantif qPCR (Roche kit ) versus standard WHO Control + = Run1

6. Genomic stability The sequence of the input (run 0)and of the viral progeny at run 5 are IDENTICAL C39 : 5594 bp sequenced 99,3 % identity with stain HV (Genbank)

7. Cells + Anti-globoside Ab 1 hour 4°C B19 Cells 2 hour 4°C Washing 3x 48 hours 37°C Supernatant DNA Extraction Nested-PCR ? Specific Inhibition of B19 infectivity by anti-receptor (globoside)

8. 23 B19 48 hours at 37°C HepG2 B19 Neutralisation by specific anti-VP2 capsid IgG Anti-capsid ANTIBODIES + 16 hours at RT Culture Supernatant DNA extraction NESTED PCR Washing 3x ? HepG2

9. B19 : C39 positive donation devoid of anti-B19 IgG or IgM Multiplicity of infection (MOI) : 100 IU/2 10 5 cells. Detection by end-point dilution and Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258) Inhibition of B19 infectivity by specific anti-VP2 capsid protein antibodies

10. Measure of infectivity : an assay more sensitive than qPCR! Minimal infectious dose : 0,1 IU Input 0,1 IU gives a viral progeny 1 IU is more than 10 infectious particles => 1 IU is more than 10 infectious particles Detectable end-point (log dilution)

11. Concentration of IVIG to obtain 50% virus neutralisation - 10 ng/ml for IVIG 2 ( MULTIGAM) - 300 ng/ml for IVIG 1 ( SANDOGLOBULIN) 0 25 50 75 100 -4-2024 Log Human IgG (µg/ml) INHIBITION (%) NIBSC IVIG 1 IVIG 2 Method: -B19 DNA (10 3 IU) from a single plasma donation -Incubation overnight at room temperature with IVIG at concentrations 3x10 -4 to 300 µg/ml Validation of IVIG neutralisation capacity (1) Applications

12. ProductBatchReduction factor (log) Nanogam1 >4.15 Nanogam2 >3.81 Nanogam3 >3.95 Nanogam4 >4.02 Gammaquin1 >5.32 Gammaquin2 >5.36 Gammaquin3 >5.23 Validation of IVIG neutralisation capacity (2)

13. DONOR 05 B19 multiplication +++------- Each sample was diluted to obtain 1000 IU B19-DNA before infecting the cells. Applications Measure B19 infectivity from donor plasma

14. Applications Validation of UVC efficiency for inactivation of B19 B19 (C39) is inoculated into HepG2 cultures. The supernatant containing B19 (1st round) is added to fresh cells (2nd round). UVC induces defective viruses 2

15. Conclusions HepG2 cells efficiently produce infectious B19 virus (5 successive runs) The sequence of the viral progeny is identical to the input : genomic stability Infectivity assay highly sensitive : 0,1 IU of B19 gives a viral progeny Excellent tool for B19 virus validations

16. ULB Op de beeck A. Draps M.-L. Caillet-Fauquet P. CAF-DCF Branckaert Th. Baurin S. Timmerman D. Laub R. German Red Cross Schmidt M. Sanquin Over J. Sanquin Oye Tolo H.