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Active B19 virions production in hepatoblastoma and hepatocarcinoma cell lines: amplification and genomic stability.

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

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Active B19 virions production in hepatoblastoma and hepatocarcinoma cell lines: amplification and genomic stability.

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  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 Brussels1. • R&D- Central Department for Fractionation, Brussels2

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

  4. A 0.1 IU 10 IU 100 IU 0 IU A 0.1 IU 10 IU 100 IU 0 IU HepG2 HuH7 7 7 6 6 5 5 Detectable end-point Detectable end-point (log dilution) 4 (log dilution) 4 3 3 2 2 1 1 0 0 24 48 72 24 48 72 Post infection time (h) Post infection time (h) Detection of B19 in the supernatantof HepG2 and Huh-7 • B19 : plasma WHO 99/800 • Multiplicity of infection (MOI) : 0.1-100 IU/5 105 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)

  5. Viral progeny is infectious B19 : C39 positive donation devoid of anti-B19 IgG or IgM Input of each run : 100 IU/ 5 105 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. ? Specific Inhibition of B19 infectivity by anti-receptor (globoside) 2 hour 4°C B19 1 hour 4°C + Cells Cells Washing 3x Anti-globoside Ab 48 hours 37°C DNA Extraction Nested-PCR Supernatant

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

  9. Inhibition of B19 infectivity by specific anti-VP2 capsid protein antibodies • B19 : C39 positive donation devoid of anti-B19 IgG or IgM • Multiplicity of infection (MOI) : 100 IU/2 105 cells. • Detection by end-point dilution and Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)

  10. Detectable end-point (log dilution) 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

  11. Applications 100 75 INHIBITION (%) NIBSC 50 IVIG 1 25 IVIG 2 0 -4 -2 0 2 4 Log Human IgG (µg/ml) Validation of IVIG neutralisation capacity (1) Concentration of IVIG to obtain 50% virus neutralisation - 10 ng/ml for IVIG 2 (MULTIGAM) - 300 ng/ml for IVIG 1 (SANDOGLOBULIN) Method: - B19 DNA (103 IU) from a single plasma donation - Incubation overnight at room temperature with IVIG at concentrations 3x10-4 to 300 µg/ml

  12. Applications Validation of IVIG neutralisation capacity (2)

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

  14. 2 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

  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. Sanquin Over J. Sanquin Oye Tolo H. German Red Cross Schmidt M.

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