Perrine Caillet-Fauquet

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

• Predilection for infecting dividing cells
• Low genetic complexity of B19 links its
  replication highly to cell factors (S phase and
  differentiation)
• 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 (b integrin)

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• Primary cultures
• Bone marrow cells (human-monkey)
• CFU-e peripheral blood
• Umbilical cord blood cells
• Fetal liver cells

• Cell lines
• Blast cell lines UT-7
• TF-1
• M-07
• B1647
• Megacaryotic leukemia cell line MB-02
• Megacaryotic JK-1
• Erythroid cell line KU812
• Erythroid cell line KU812 Ep6
• Erythroid cell line KU812F in hypoxia (6O2)
• Hepatocarcinoma cell lines

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KU812F pluripotent cells
Elimination of the remaining input and 3X
wash addition of medium
B19 Virus (WHO 99/800, NISBC )
INCUBATION 3 days
INCUBATION 2 hours
Infected cells
KU812F
CENTRIFUGATION
6O2 or 20O2
6O2 or 20O2
EPO
CENTRIFUGATION
Supernatant DNA extraction Nested-PCR
cells
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B19 production in KU812F cells (epo) Hypoxia
compared to Normoxia
3
2
B19 detectable end-point
(log dilution)
1
0
0
1
10
100
1000
10000
Virus input (IU)
6O2 20O2
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HepG2(or HuH7) Cellular model for B19 production
WHO 99/800, NISBC
B19
2 hours 37C
Cells
Cells
Washing 3x Centrifugation
HepG2 is Human hepatoblastoma cell line
24, 48, 72 hours 37C
Cells
DNA Extraction PCR Amplification
PCR POSITIVE
Supernatant
Erythrovirus B19
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B19 production in HepG2 cells and HuH7
0.1 IU
10 IU
100 IU
0 IU
A
HepG2
7
6
5
(log dilution)
Detectable end-point
4
3
2
1
0
24
48
72
Post infection time (h)
0.1 IU
10 IU
100 IU
0 IU
A
HuH7
7
6
5
Detectable end-point
(log dilution)
4
3
2
1
0
24
48
72
Post infection time (h)
8
Measure of apotosis annexin V positive
hepatocarcinoma cells
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B19 transcription in HepG2
72h
48h
24h
RT-PCRspliced mRNA VP
183bp
P6
100
0
Map units
NS1
B19 input IU
102
1
1
102
1
102
Hep NI
Hep NI
VP1
VP1
72h
48h
24h
VP2
VP2
Actin
Protein 11 kDa
B19L212082-2067 B19L202066-2050
B19L2381-404 (Brunstein et al 2000)
Unspliced 1702 Splice donor 406 splice acceptors
1910 183bp amplicon sizes
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Inhibition of B19 infection in HepG2 by
antibodies anti-P blood group antigen
2 hour 4C
B19
1 hour 4C

Cells
Cells
Washing 3x
Anti-P
48 hours 37C
DNA Extraction Nested-PCR
Supernatant
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B19 Neutralization
B19
ANTIBODIES
16 hours at RT

48 hours at 37C
Washing 3x
Culture Supernatant
?
HepG2
HepG2
DNA extraction NESTED PCR
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RABBIT MODEL OF ANTI-B19 NEUTRALIZING ANTIBODIES

• Method
• Selection of potential VP/NS epitopes
• Rabbit immunization
• Purification of rabbit IgG
• Elisa on peptides and Commercial Kit (Biotrin)
• Testing in the cellular model

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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 (103 IU) from a single plasma donation
• incubation Over Night at room temperature with
• IVIG concentrations 3x10-4 to 300 µg/ml

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• Conclusions
• New assay for parvovirus B19 infectivity and
  neutralization.
• Use of B19 as relevant model for virus
  inactivation methods .

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