Hepatitis C Virus

Hepatitis C Virus Inactivation
PD Dr. Eike Steinmann
Centre for Experimental and Clinical Infection Research
Institute of Experimental Virology, Twincore*
*joint venture between Medical School Hannover and Helmholtz Centre for Infection Research
Hepatitis C Virus (HCV) Profile
Family:
Flaviviridae
Genus:
Hepacivirus
Species:
Hepatitis C virus
(7 genotypes)
Size:
50-60 nm
Genome:
(+) ssRNA, ~9.6 kb
Prevalence:
160 million patients
Therapy:
next talk
E2
E1
ss(+) RNA
Nucleocapsid (core)
Lipid membrane
Ways of HCV Transmissions
medical procedures
drug abuse
sex 15%
i.v. drug abuse 60%
transfusions 10%
unknown
10%
transfusion
sex
vertical
other
Virucidal testing using surrogate viruses
testvirus
surrogate-virus
Hepatitis B Virus
Duck hepatitis B virus (DHBV)
Norovirus
feline Calicivirus (FCV)
murine Norovirus (MNV)
Hepatitis C Virus
bovine viral diarrhea virus
(BVDV)
HCV infection system
In vitro Transcription
T7-Polymerase
Plasmid
T7-Promotor
NTR
Core
E1
E2
amp
NTR
NS3
NS5B
HCV RNA
NS5A NS4B
transfection
96 h
p7
NS2
pFK-JFH1
NS4A
Detection infected cells
(IFM, qRT-PCR)
72 h
106
Huh7 hepatoma cells
IFM
105
104
103
ΔE1-E2
Wakita and Pietschmann et al., Nat Med 2005
JFH1/wt
α – NS3
102
HCV RNA copies/well
HCV RNA transcriptes
Properties of cell culture-grown HCV particles
• Density peaks in sucrose gradient ca. 1.15 g/ml
E2 - Mab
• Spherical particles with diameter of ca. 55 nm
JFH1 wildtype
• Infectivity can be neutralized by CD81-specific antibodies
• Infectivity can be neutralized by Ig from patients and anti-E2 Mab
• Cell culture-grown HCV is infectious in vivo (chimp; chimeric mouse)
Virucidal efficacy of different alcohols against HCV
Quantitative suspension:
100 µl virus
900 µl disinfectant
mix, incubation, mix
titration infectivity (TCID50/ml)
Effect of ethanol, 1-propanol and 2-propanol on HCV/BVDV
Log10 infectivity TCID50/ml
HCV
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
0
contr.
5%
10%
20%
30%
40%
ethanol
1-propanol
2-propanol
detection limit
contr.
5%
10%
20%
30%
40%
incubation time 5 min
incubation time 1 min
Log10 infectivity TCID50/ml
BVDV
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
ethanol
1-propanol
2-propanol
0
contr.
5%
10%
20%
incubation time 1 min
30%
40%
contr.
5%
10%
20%
incubation time 5 min
30%
40%
HCV infectivity in comparison with HCV-RNA copy numbers
Log10 infectivity TCID50/ml
HCV
21°C
0, 7, 14, 28, 35 days
infectivity
9
8
7
6
5
4
3
2
1
0
0
7
14
21
28
35
28
35
days
titration infectivity (TCID50/ml)
RNA isolation and quantification (qRT-PCR)
RNA 0 days
RNA 21 days
Log10 RNA copies/ml
RNA copies (3x107)
RNA copies
11
10
9
8
7
6
0
7
14
days
21
HCV stability and inactivation in suspension
7
HCV+Serum
Log RLU/well
6
5
4
3
2
0
7
14
days
21
28
Log10 infectivity TCID50/ml
HCV
7
HCV
6
cytotoxicity
5
4
3
2
1
0
contr. 1
2
3
4
5
6
7
undiluted, 30 sec
Ciesek et al. Journal of Infectious Diseases 2010
Steinmann et al. American Journal of Infection Control 2010
Steinmann et al. Antimicrobial Resistance and Infection Control 2013
Establishment of a HCV carrier assay
virusinoculum
50 µL
(drying)
Log10 Infectivity TCID50/ml
Log10 Infectivity TCID50/ml
900 µL
medium Ø FCS
(1 min vortex)
(incubation for 1 min)
titration infectivity
5
7
6
5
4
3
2
1
0
100 µL
test substance
suspension
dried
4
3
2
1
0
Control 10
20
30
40
50
1-propanol
60
10
20
30
40
50
2-propanol
60
10
20
30
40
50
60%
ethanol
Dörrbecker et al. Journal of Infectious Diseases 2011
Survival of dried HCV on inanimate surfaces
Log10 Infectivity TCID50/ml
6
HCV
cytotoxicity
5
4
3
2
1
0
Control 0.025 0.25
0.50 0.025 0.25
C
B
A
5
Log10 Infectivity TCID50/ml
0.50 0.025 0.25
HCV - serum
HCV + serum
4
3
2
1
0
0
1
2
3
4
days
5
6
7
8
0.50 0.025 0.25
D
0.50%
Development of a drug transmission assay
5
Log10 RLU/well
4
3
2
1
0
15
25
35
45
55
65
temperature in °C
75
In collaboration with NDRI, NY, USA Dr. Mateu-Gelabert, Prof. Hagan, Prof. Des Jarlais and Fixpunkt Hannover
Transmission of Hepatitis C virus among injecting drug users: viral
stability and association with drug preparation equipment
4
500 µl
3
200 µl
40 µl
2
8 µl
1.5 µl
1
0
0
1
2
4
6
8
10 12
days
14
16
18
20
5
virus spike
Log10 Infectivity TCID50/ml
Log10 Infectivity TCID50/ml
5
recovery concentrated
4
3
2
1
0
plastic
aluminium
glass
container material
Dörrbecker et al. Journal of Infectious Diseases 2013
Transmission of Hepatitis C virus among injecting drug users: viral
stability and association with drug preparation equipment
Dörrbecker et al. Journal of Infectious Diseases 2013
HCV transmission by anaesthetica
6
control
propofol
Log10 infectivity ffu/ml
5
4
3
2
1
0
7
14
21
days
28
35
Gutelius et al. Gastroenterology 2010
Fischer et al. Clinical Infectious Diseases 2010
Steinmann et al. Clinical Infectious Diseases 2011
Behrendt et al. American Journal of Infection Control 2013
Inactivation of HCV in blood products
Steinmann et al. Transfusion 2012
Inactivation kinetics of HCV in plasma by MB plus light treatment
Steinmann et al. Transfusion 2012
Inactivation kinetics of HCV by UVC irradiation of Platelet Concentrates
Steinmann et al. Transfusion 2012
Thermo-stability of seven Hepatitis C virus genotypes in vitro and in vivo
1b
2a
3a
4a
5a
6a
7a
9
8
7
6
5
4
3
2
1
0
0
7
14
21
28
35
time [days]
Log10 HCV RNA [IU/mL]
Log10 Infectivity [TCID50/ml]
1a
10
1a
1b
2a
3a
4a
5a
6a
7a
pool
Dörrbecker et al. Journal of Viral Hepatitis 2013
Inactivation of HCV in human mother’s milk
Pfaender et al. Journal of Infectious Diseases 2013
Acknowledgement
Twincore, Institute of Experimental Virology
Dorothea Bankwitz
Pathrick Behrendt
Richard Brown
Janina Brüning
Patrick Chhatwal Thomas Pietschmann Sandra Ciesek Martina Friesland Juliane Dörrbecker Nina Riebesehl Corinne Wilhelm
Anne Frentzen
Gisa Gerold
Sabine Giese
Cooperations
Christina Grethe
T. v. Hahn, M.P. Manns, H. Wedemeyer
Sibylle Haid
Joerg Steinmann
Essen, Germany
Kathrin Hüging
Philip Meuleman
Ghent, Belgium
Luis Schang, Che Colpitts
Edmonton, Canada
Peter Friebe
Berkeley, USA
Michael Diamond
St Louis, USA
Mandy Klaske
Angga Kusuma
Paula Perin
Stephanie Pfänder
Wiebke Rastedt
Gabrielle Vieyres
Stephanie Walter
Kathrin Welsch
Hannover, Germany
Reagents:
C. Rice
New York, USA
D. Moradpour
Lausanne, Switzerland
Funding: