title: Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies.
abstract: Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have not been studied at the single-cell level, leaving a gap in our understanding of antiviral kinetics and host-virus interactions. Here, we demonstrate quantitative imaging of HCV genomes in multiple infection models, and multiplexing of positive and negative strand vRNAs and host antiviral RNAs. We capture the varying kinetics with which antiviral drugs with different mechanisms of action clear HCV infection, finding the NS5A inhibitor daclatasvir to induce a rapid decline in negative-strand viral RNAs. We also find that the induction of host antiviral genes upon interferon treatment is positively correlated with viral load in single cells. This study adds smFISH to the toolbox available for analyzing the treatment of RNA virus infections.
authors.affiliation: Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Vyas
authors.initials: V
authors.lastname: Ramanan
authors.affiliation: Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kartik
authors.initials: K
authors.lastname: Trehan
authors.affiliation: Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Mei-Lyn
authors.initials: ML
authors.lastname: Ong
authors.affiliation: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, 10065 NY, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Luna
authors.affiliation: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, 10065 NY, USA.
authors.forename: Hans-Heinrich
authors.initials: HH
authors.lastname: Hoffmann
authors.affiliation: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, 10065 NY, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Espiritu
authors.affiliation: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, 10065 NY, USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Sheahan
authors.affiliation: Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Hamsika
authors.initials: H
authors.lastname: Chandrasekar
authors.affiliation: Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Schwartz
authors.affiliation: Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Christine
authors.affiliation: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, 10065 NY, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: van Oudenaarden
authors.affiliation: Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Division of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: sbhatia@mit.edu.
authors.forename: Sangeeta N
authors.initials: SN
authors.lastname: Bhatia
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D016679:Genome, Viral; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000821:virology*; D054884:Host-Pathogen Interactions / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D012367:RNA, Viral; D059010:Single-Cell Analysis / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins
keywords: HCV; Host–virus interactions; Single-molecule FISH; Viral replication