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
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Op de beeck A. 1 , Draps M.-L. 1 , Baurin S. 2 , Timmerman D. 2 ,
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 - PowerPoint PPT Presentation
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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
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)
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°CCells
Washing 3x
24, 48, 72 hours
37°C
Supernatant PCRPOSITIVE
DNA Extraction
PCR Amplification
Cells
Cells
WHO 99/800, NIBSC
Caillet-Fauquet et al, Transfusion 2004; 44:1340-3.
Detection of B19 in the supernatant of HepG2 and Huh-7
B19 : plasma WHO 99/800Multiplicity of infection (MOI) : 0.1-100 IU/5 105 cells : low M.O.I. ! Minimal infectious dose : 0.1 to 1 IU in HepG2Detection by Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Det
ecta
ble
en
d-p
oin
t (l
og
dil
uti
on
)
0.1 IU 10 IU 100 IU 0 IUA HepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Det
ecta
ble
en
d-p
oin
t (l
og
dil
uti
on
)
0.1 IU 10 IU 100 IU 0 IUA HuH7
Viral progeny is infectious
B19 : C39 positive donation devoid of anti-B19 IgG or IgMInput of each run : 100 IU/ 5 105 cells (m.o.i. = 0.002) Quantif qPCR (Roche kit ) versus standard WHOControl + = Run1
Genomic stabilityThe sequence of the input (run 0)and of the viral progeny
at run 5 are IDENTICAL
C39 : 5594 bp sequenced99,3 % identity with stain HV (Genbank)
Cells +
Anti-globoside Ab
1 hour
4°C
B19
Cells
2 hour4°C
Washing 3x
48 hours37°C
Supernatant
DNA ExtractionNested-PCR
?
0
1
2
3
4
5
CONTROL + ANTI-P
Detectable end-point
(log dilution)
HepG2
HepG2
HuH7
HuH7
Specific Inhibition of B19 infectivity by anti-receptor (globoside)
23
B19
48 hours at 37°C
HepG2
B19 B19 NeutralisationNeutralisation by specific by specific anti-VP2 capsid IgGanti-VP2 capsid IgG
Anti-capsid ANTIBODIES
+16 hours
at RT
Culture Supernatant
DNA extractionNESTED PCR
Washing 3x?
HepG2
0
25
50
75
100
-5 -4 -3 -2 -1 0 1 2
Log rabbit IgG (µg/ml)
Inhibition (%)
Ser 48 - Ser 57
Ser 285 - Lys 300
Ser 554 - Tyr 572
Lys 720 - His 740
Control Peptide
B19 : C39 positive donation devoid of anti-B19 IgG or IgMMultiplicity 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)
Inhibition of B19 infectivity by specific anti-VP2 capsid protein antibodies
Measure of infectivity : an assay more sensitive than qPCR!
Minimal infectious dose : 0,1 IUInput 0,1 IU gives a viral progeny
=> 1 IU is more than 10 infectious particles1 IU is more than 10 infectious particles
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
Det
ecta
b le
end-
poin
t (lo
g d i
lutio
n)
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 -2 0 2 4
Log Human IgG (µg/ml)
INH
IBIT
ION
(%
)
NIBSC
IVIG 1
IVIG 2
Method:- B19 DNA (103 IU) from a single plasma donation - Incubation overnight at room temperature with