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Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system

Abstract

Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.

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Figure 1: An IPS-1-based reporter system for detection of HCV infection.
Figure 2: Time-lapse live-cell imaging of HCVcc infection.
Figure 3: Use of the IPS-1-based reporter to expand HCVcc culture systems.

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Acknowledgements

We acknowledge the expert support of The Rockefeller University Bioimaging Core Facility, with special thanks to A. North, S. Galdeen and S. Bhuvanendran. We thank The Rockefeller University Flow Cytometry Resource Center, supported by the Empire State Stem Cell Fund through NY State Department of Health (NYSDOH) contract no. C023046; opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Fund, the NYSDOH, or the State of NY. We are grateful to C. Stoyanov (The Rockefeller University) for YF17D(5′C25Venus2AUbi), J. Tazi for G3BP (Institut de Génétique Moléculaire de Montpellier) and I. Frolov (UTMB) for Venezuelan equine encephalitis virus-EGFP. We thank M. Holz, A. Forest, M. Panis and A. Webson for laboratory support and C. Murray for critical reading of the manuscript. This work was supported by Public Health Service grants R01 AI075099 (C.M.R.) and R01 DK56966 (S.N.B.). This work was also funded by the Office of the Director/National Institutes of Health (NIH) through the NIH Roadmap for Medical Research, Grant 1 R01 DK085713-01 (C.M.R. and S.N.B.). Information on this Roadmap Transformative R01 Program can be found at http://grants.nih.gov/grants/guide/rfa-files/RFA-RM-08-029.html. Additional funding was provided by the Greenberg Medical Research Institute and the Starr Foundation (C.M.R.). S.N.B. is an Howard Hughes Medical Investigator investigator. C.T.J. was supported by National Research Service Award DK081193; M.T.C. was supported by a Women & Science Fellowship; L.M.J.L. is supported by a Natural Sciences and Engineering Research Council of Canada fellowship. A.P. is a recipient of a Kimberly Lawrence-Netter cancer research discovery fund award.

Author information

Authors and Affiliations

Authors

Contributions

C.T.J. and C.M.R. designed the project, analyzed results and wrote the manuscript. C.T.J., M.T.C., L.M.J.L., A.J.S., S.R.K., T.S.O., A.P. and J.W.S. performed the experimental work. S.R.K., J.W.S., T.S.O., M.R.M. and S.N.B. contributed reagents and technical expertise.

Corresponding author

Correspondence to Charles M Rice.

Ethics declarations

Competing interests

The authors declare the following conflicts of interest, which are managed under University policy: C.M.R. has equity in Apath, LLC, which holds commercial licenses for the Huh-7.5 cell line and the HCVcc cell culture system. S.R.K and S.N.B have equity in Hepregen Corporation, which holds commercial licenses from the Massachusetts Institute of Technology for micropatterned co-cultures (MPCCs).

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–3 (PDF 730 kb)

Supplementary Video 1a

Supplementary Video 1. Time-lapse live cell imaging of HCVcc infection. (MOV 834 kb)

Supplementary Video 1b

Supplementary Video 1. Time-lapse live cell imaging of HCVcc infection. (MOV 1057 kb)

Supplementary Video 1c

Supplementary Video 1. Time-lapse live cell imaging of HCVcc infection. (MOV 510 kb)

Supplementary Video 1d

Supplementary Video 1. Time-lapse live cell imaging of HCVcc infection. (MOV 629 kb)

Supplementary Video 2a

Supplementary Video 2. Time-lapse live cell imaging of HCV-induced stress response. (MOV 1837 kb)

Supplementary Video 2b

Supplementary Video 2. Time-lapse live cell imaging of HCV-induced stress response. (MOV 3523 kb)

Supplementary Video 2c

Supplementary Video 2. Time-lapse live cell imaging of HCV-induced stress response. (MOV 4147 kb)

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Jones, C., Catanese, M., Law, L. et al. Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system. Nat Biotechnol 28, 167–171 (2010). https://doi.org/10.1038/nbt.1604

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