title: A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening.
abstract: Hepatitis C virus (HCV) is a significant human pathogen infecting 3% of the world population. An infectious molecular clone capable of replicating and releasing infectious virions in cell culture has only been available since 2005, leaving a significant knowledge gap concerning post-RNA replication events such as particle assembly, trafficking and release. Thus, a fast, efficient and accurate method of measuring infectious viral titres is highly desirable. Current methods rely upon manual counting of infected cell foci and so are both labour-intensive and susceptible to human error. Here, we report a novel protocol, which utilises the IncuCyte ZOOM instrument and related software to accurately count infected cells and extrapolation of this data to produce an infectious titre, reported as infectious units per millilitre (IU/mL). This method reduces cost, time and error in experiments. We also demonstrate that this approach is amenable to high-throughput compound screening, thereby expediting the identification of novel antivirals.
authors: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors: Hazel
authors: H
authors: Stewart
authors: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors: Christopher
authors: C
authors: Bartlett
authors: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors: Douglas
authors: D
authors: Ross-Thriepland
authors: Leeds Institute of Cancer and Pathology, Faculty of Medicine and Health, St James's University Hospital, University of Leeds, Leeds LS9 7TF, United Kingdom.
authors: Joseph
authors: J
authors: Shaw
authors: Leeds Institute of Cancer and Pathology, Faculty of Medicine and Health, St James's University Hospital, University of Leeds, Leeds LS9 7TF, United Kingdom.
authors: Stephen
authors: S
authors: Griffin
authors: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. Electronic address: M.Harris@leeds.ac.uk.
authors: Mark
authors: M
authors: Harris
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D016679:Genome, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D011759:Pyrrolidines; D012367:RNA, Viral / Q000235:genetics; D000069474:Sofosbuvir / Q000494:pharmacology; D014633:Valine / Q000031:analogs & derivatives; D019562:Viral Load / Q000379:methods*; D014779:Virus Replication / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D011759:Pyrrolidines; D012367:RNA, Viral; D014633:Valine; C549273:daclatasvir; D000069474:Sofosbuvir
keywords: Antivirals; Diagnosis; Hepatitis C virus; IncuCyte ZOOM; Titration