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: Genus: Flaviviridae Hepacivirus E E Species: Hepatitis C virus (7 genotypes) Size: - nm Genome: (+) ssrna, ~9. kb Prevalence: Therapy: million patients next talk ss(+) RNA Nucleocapsid (core) Lipid membrane
Ways of HCV Transmissions drug abuse medical procedures i.v. drug abuse % sex % transfusions % unknown % 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 JFH/wt ΔE-E HCV RNA copies/well HCV RNA transcriptes In vitro Transcription T7-Polymerase amp NTR T7-Promotor Plasmid NTR Core E pfk-jfh E p7 NS HCV RNA NSB NSA NSA NSB NS transfection Detection infected cells (IFM, qrt-pcr) 9 h 7 h Huh7 hepatoma cells IFM Wakita and Pietschmann et al., Nat Med α NS
E - Mab Properties of cell culture-grown HCV particles Density peaks in sucrose gradient ca.. g/ml Spherical particles with diameter of ca. nm JFH wildtype Infectivity can be neutralized by CD8-specific antibodies Infectivity can be neutralized by Ig from patients and anti-e Mab Cell culture-grown HCV is infectious in vivo (chimp; chimeric mouse)
Virucidal efficacy of different alcohols against HCV Quantitative suspension: µl virus 9 µl disinfectant mix, incubation, mix titration infectivity (TCID /ml)
Log infectivity TCID /ml Log infectivity TCID /ml Effect of ethanol, -propanol and -propanol on HCV/BVDV HCV 8 7 8 7 ethanol -propanol -propanol contr. % % % % % contr. % % % % % detection limit incubation time min incubation time min BVDV 8 7 8 7 ethanol -propanol -propanol contr. % % % % % contr. % % % % % incubation time min incubation time min
Log RNA copies/ml Log infectivity TCID /ml HCV infectivity in comparison with HCV-RNA copy numbers HCV C, 7,, 8, days titration infectivity (TCID /ml) RNA isolation and quantification (qrt-pcr) 9 8 7 infectivity 7 8 days RNA copies RNA copies (x 7) RNA days RNA days 9 8 7 7 8 days
Log RLU/well Log infectivity TCID /ml HCV stability and inactivation in suspension 7 HCV 7 HCV HCV+Serum cytotoxicity 7 8 contr. 7 days undiluted, sec Ciesek et al. Journal of Infectious Diseases Steinmann et al. American Journal of Infection Control Steinmann et al. Antimicrobial Resistance and Infection Control
Log Infectivity TCID /ml Log Infectivity TCID /ml Establishment of a HCV carrier assay virusinoculum µl (drying) µl test substance 9 µl medium Ø FCS ( min vortex) (incubation for min) titration infectivity 7 suspension dried Control -propanol % -propanol ethanol Dörrbecker et al. Journal of Infectious Diseases
Log Infectivity TCID /ml Log Infectivity TCID /ml Survival of dried HCV on inanimate surfaces HCV cytotoxicity Control............% A B C D HCV - serum HCV + serum 7 8 days
Development of a drug transmission assay Log RLU/well 7 temperature in C In collaboration with NDRI, NY, USA Dr. Mateu-Gelabert, Prof. Hagan, Prof. Des Jarlais and Fixpunkt Hannover
Log Infectivity TCID /ml Log Infectivity TCID /ml Transmission of Hepatitis C virus among injecting drug users: viral stability and association with drug preparation equipment µl µl µl 8 µl. µl 8 8 days virus spike recovery concentrated plastic aluminium glass container material Dörrbecker et al. Journal of Infectious Diseases
Transmission of Hepatitis C virus among injecting drug users: viral stability and association with drug preparation equipment Dörrbecker et al. Journal of Infectious Diseases
Log infectivity ffu/ml HCV transmission by anaesthetica control propofol 7 8 days Gutelius et al. Gastroenterology Fischer et al. Clinical Infectious Diseases Steinmann et al. Clinical Infectious Diseases Behrendt et al. American Journal of Infection Control
Inactivation of HCV in blood products Steinmann et al. Transfusion
Inactivation kinetics of HCV in plasma by MB plus light treatment Steinmann et al. Transfusion
Inactivation kinetics of HCV by UVC irradiation of Platelet Concentrates Steinmann et al. Transfusion
Thermo-stability of seven Hepatitis C virus genotypes in vitro and in vivo Log Infectivity [TCID /ml] Log HCV RNA [IU/mL] a b a a a a a 7a 9 8 7 7 8 time [days] a b a a a a a 7a pool Dörrbecker et al. Journal of Viral Hepatitis
Inactivation of HCV in human mother s milk Pfaender et al. Journal of Infectious Diseases
Acknowledgement Twincore, Institute of Experimental Virology Dorothea Bankwitz Pathrick Behrendt Richard Brown Janina Brüning Patrick Chhatwal Anne Frentzen Gisa Gerold Sabine Giese Christina Grethe Sibylle Haid Kathrin Hüging Mandy Klaske Angga Kusuma Paula Perin Stephanie Pfänder Wiebke Rastedt Gabrielle Vieyres Stephanie Walter Kathrin Welsch Thomas Pietschmann Sandra Ciesek Martina Friesland Juliane Dörrbecker Nina Riebesehl Corinne Wilhelm Cooperations T. v. Hahn, M.P. Manns, H. Wedemeyer Hannover, Germany Joerg Steinmann Essen, Germany Philip Meuleman Ghent, Belgium Luis Schang, Che Colpitts Edmonton, Canada Peter Friebe Berkeley, USA Michael Diamond St Louis, USA Reagents: C. Rice New York, USA D. Moradpour Lausanne, Switzerland Funding: