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

Nature Biotechnology volume 28, pages 167–171 (2010)Cite this article

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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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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.

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Author notes

  1. Salman R Khetani & Andrew J Syder
    Present address: Present addresses: Hepregen Corporation, Medford, Massachusetts, USA (S.R.K.) and iTherX Pharmaceuticals, San Diego, California, USA (A.J.S.).,

Authors and Affiliations

  1. Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA
    Christopher T Jones, Maria Teresa Catanese, Lok Man J Law, Andrew J Syder, Alexander Ploss, Thomas S Oh, John W Schoggins, Margaret R MacDonald & Charles M Rice
  2. Division of Health Sciences and Technology, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
    Salman R Khetani & Sangeeta N Bhatia
  3. Howard Hughes Medical Institute, Boston, Massachusetts, USA
    Sangeeta N Bhatia
  4. Division of Medicine, Brigham & Women's Hospital, Boston, Massachusetts, USA
    Sangeeta N Bhatia

Authors

  1. Christopher T Jones
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  2. Maria Teresa Catanese
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  3. Lok Man J Law
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  4. Salman R Khetani
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  5. Andrew J Syder
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  6. Alexander Ploss
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  7. Thomas S Oh
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  8. John W Schoggins
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  9. Margaret R MacDonald
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  10. Sangeeta N Bhatia
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  11. Charles M Rice
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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 toCharles M Rice.

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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).

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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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