∆E1-E2HCV RNA copies/wellJFH1/wt

Inaktivierung von HCV ^{und HIV}

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

Family: Flaviviridae Genus: Hepacivirus

Species: Hepatitis C virus (7 genotypes)

Size: 50-60 nm

Genome: (+) ssRNA, ~9.6 kb Prevalence: 160 million patients Therapy: PEG-IFN Riba, DAAs

Hepatitis C Virus (HCV) Profile

E2 E1

Lipid membrane

Nucleocapsid (core)

ss(+) RNA

Ways of HCV Transmissions

drug abuse medical procedures

i.v. drug abuse 60%

sex 15%

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)

96 h

transfection Detection infected cells

(IFM, qRT-PCR)

Huh7 hepatoma cells HCV RNA

72 h

α – NS3 IFM

∆E1-E2

10² 10³ 10⁴ 10⁵ 10⁶

HCV RNA copies/well

JFH1/wt

HCV RNA transcriptes

T7-Polymerase

In vitro Transcription Plasmid

amp

Core E1

E2 p7

NS2

NS3 NS4A NS4B NS5A NS5B

T7-Promotor

NTR

NTR

pFK-JFH1

HCV infection system

Wakita and Pietschmann et al., Nat Med 2005

Virucidal efficacy of different alcohols against HCV

100 µl virus 900 µl disinfectant

mix, incubation, mix

titration infectivity (TCID

/ml)

Quantitative suspension:

ethanol 1-propanol

2-propanol

incubation time 1 min 0

1 2 3 4 5 6 7 8

contr. 5% 10% 20% 30% 40%

Log10infectivity TCID50/ml

contr. 5% 10% 20% 30% 40%

0 1 2 3 4 5 6 7 8

incubation time 5 min

Effect of ethanol, 1‐propanol and 2‐propanol on HCV/BVDV

HCV

Log10infectivity TCID50/ml 0 1 2 3 4 5 6 7 8

contr. 5% 10% 20% 30% 40% 0 contr. 5% 10% 20% 30% 40%

1 2 3 4 5 6 7 8

incubation time 1 min incubation time 5 min

ethanol 1-propanol

2-propanol

BVDV

detection limit

Ciesek et al. Journal of Infectious Diseases 2010

HCV stability and inactivation in suspension

Log RLU/well

days 2

3 4 5 6 7

0 7 14 21 28

HCV

HCV+Serum

0 1 2 3 4 5 6 7

contr. 1 2 3 4 5 6 7

HCV

cytotoxicity

undiluted, 30 sec Log10infectivity TCID50/ml

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 2014

Effect of different disinfectants against HCV 

formaldehyde 1 min w/o column

0 1 2 3 4 5 6 7 8

vc 0,01% 0,07% 0,7%

Log10 Infectivity TCID50/ml

HCV

cytotoxicity

<1500 <1500

<15000

<15

peracetic acid 1min w/o column

0 1 2 3 4 5 6 7 8

0,001% 0,05% 0,1%

Log10 Infectivity TCID50/ml

HCV

cytotoxicity

<15000

<1500

<15 <15

glutaraldehyde 1 min w/o column

0 1 2 3 4 5 6 7 8

vc 0,01% 0,1% 0,5%

Log10 Infectivity TCID50/ml

HCV

cytotoxicity

<1500

<150

<150

<15

Infectivity TCID50/ml

1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06

virus control

(H2O) virus control

(PBS) Tween

0,1% Tween

0,5%

Inactivation of HCV: Triton‐X and paraformaldehyde

cytotoxicity

virus cont Triton X 0,1%

1min

virus titer

Triton X 0,5%

1min 1,00E+00

1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06

Triton X 0,05%

1min

Triton X 0,05%

3min

Triton X 0,05%

5min

Infectivity TCID50/ml

1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06

virus control   2,7% pfa 10min

0,3% pfa 10min virus titer

InfectivityTCID50/ml

cytotoxicity

Mechanisms of viral inactivation methods against hepatitis C virus

E2 E1

RNA Core Lipid membrane

• Triton

• Ethanol

• 2‐Propanol

• PVP‐I

• Heat

• UV

Pfaender et al. Applied and Environmental Microbiology 2015

Mechanisms of hepatitis C virus inactivation

virusinoculum 50 µL

(drying)

titration infectivity 900 µL medium Ø FCS

(1 min vortex) 100 µL

test substance

(incubation for 1 min)

Establishment of a HCV carrier assay

Dörrbecker et al. Journal of Infectious Diseases 2011 0

1 2 3 4 5 6 7

suspension dried Log10Infectivity TCID50/ml

1-propanol 2-propanol

Control 10 20 30 40 50

Log10Infectivity TCID50/ml

10 20 30 40 50 10 20 30 40 50

60 60 60%

0 1 2 3 4 5

ethanol

Survival of dried HCV on inanimate surfaces

0 1 2 3 4 5

0 1 2 3 4 5 6 7 8

days Log10Infectivity TCID50/ml

HCV - serum HCV + serum 0

1 2 3 4 5 6

Control 0.025 0.25 0.50 0.025 0.25 0.50 0.025 0.25 0.50 0.025 0.25 0.50%

Log10Infectivity TCID50/ml

B C D

HCVcytotoxicity

A

Development of a drug transmission assay

0 1 2 3 4 5

15 25 35 45 55 65 75

temperature in °C Log10 RLU/well

Dörrbecker et al. Journal of Infectious Diseases 2011

Inactivation of HCV in blood products

1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07

Infectivity TCID50/ml

virus suspension

spike bef.

MB

0 J/cm2 after MB

10 J/cm2 20 J/cm2 40 J/cm2 120 J/cm2 Hold HCV cytotox

Mean values Blood Bag A, B, C Sample log₁₀TCID₅₀± SD log₁₀reduction factor spike bef. MB 5.41 ± 0.22

0 J/cm²after MB 5.29 ± 0.16 0.12

10 J/cm² ≤3.15 2.26

20 J/cm² ≤3.15 2.26

40 J/cm² ≤1.58 3.83

120 J/cm² ≤1.58 3.83

Mean values Blood Bag A, B, C

1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07

virus suspension

after spiking 0.05 J/cm2 0.1 J/cm2 0.2 J/cm2 0.3 J/cm2 Hold Infectivity TCID50/ml

HCV cytotox

Sample

log₁₀TCID₅₀± SD

log₁₀reduction factor

after spiking 5.61 ± 0.54

0.05 J/cm² 3.80 ± 0.12 1.81

0.1 J/cm² 2.81 ± 0.12 2.80

0.2 J/cm² ≤ 0.62 4.99

Steinmann et al. Transfusion 2012

Thermo‐stability of seven Hepatitis C virus genotypes in vitro and in vivo 

Dörrbecker et al. Journal of Viral Hepatitis 2013

0 7 14 21 28 35

Log10Infectivity [TCID50/ml]

time [days]

1a 1b 2a 3a 4a 5a 6a 7a

10 9 8 7 6 5 4 3 2 1 0

1a 1b 2a 3a 4a 5a 6a 7a pool

Log10HCV RNA [IU/mL]

Inactivation of HCV in human mother’s milk

Pfaender et al. Journal of Infectious Diseases 2013

Inactivation of HCV in human mother’s milk

Pfaender et al. Journal of Infectious Diseases 2013

Editorial Jhaveri: Protection againts HCV and other env viruses: „why breast is the best“

Inactivation of HCV and HIV by microwave!?

Inactivation of HCV and HIV by microwave

HCV

HIV

HCV/HIV

HIV/HCV

Inactivation of HCV and HIV by microwave

Acknowledgement

Twincore, Institute of Experimental Virology

Martina Friesland Juliane Dörrbecker Stephanie Pfänder Aci Siddharta Anggakusuma Daniel Todt

Michael Engelmann Stephanie Walter Svenja Drave Martinea Friesland

Christine Goffinet Thomas Pietschmann Brill & Partner

German Red Cross