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.
References
Jan 1, 1983·Differentiation; Research in Biological Diversity·A Venetianer, Z Bösze
Mar 1, 1996·Journal of Medical Virology·D MoradpourJ R Wands
Mar 12, 2002·Virus Research·Fabien Zoulim
Mar 7, 2003·The Journal of Biological Chemistry·Andrew MacdonaldMark Harris
Sep 29, 2004·Journal of Virology·Andre J MarozsanEric J Arts
Jun 11, 2005·Science·Brett D LindenbachCharles M Rice
Jun 14, 2005·Nature Medicine·Takaji WakitaT Jake Liang
Aug 17, 2005·Journal of Virology·Ania OwsiankaArvind H Patel
Feb 8, 2006·Proceedings of the National Academy of Sciences of the United States of America·MinKyung YiStanley M Lemon
May 3, 2006·Proceedings of the National Academy of Sciences of the United States of America·Thomas PietschmannRalf Bartenschlager
Mar 23, 2007·Virology·Steven G Cresawn, Richard C Condit
Apr 3, 2007·Virology Journal·Carole FournierSandrine Castelain
Jan 17, 2009·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Judith M GottweinJens Bukh
Apr 29, 2009·Antiviral Research·Michaela IroArvind H Patel
Jul 22, 2009·Antimicrobial Agents and Chemotherapy·Xuemei YuSusan L Uprichard
Feb 2, 2010·Nature Biotechnology·Christopher T JonesCharles M Rice
Sep 27, 2012·Methods : a Companion to Methods in Enzymology·Gabrielle Vieyres, Thomas Pietschmann
Nov 16, 2012·Proceedings of the National Academy of Sciences of the United States of America·Yi-Ping LiJens Bukh
Nov 23, 2012·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Khayriyyah Mohd HanafiahSteven T Wiersma
Dec 12, 2012·Journal of Virological Methods·Mahmod MuhsenAkio Fukusho
Aug 6, 2013·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Santseharay RamirezJens Bukh
Oct 4, 2013·Journal of Virology·Eike SteinmannThomas Pietschmann
Nov 23, 2013·Gastroenterology·Yi-Ping LiJens Bukh
Apr 29, 2014·Gastroenterology·David R McGivernStanley M Lemon
Aug 7, 2014·The Journal of General Virology·Sophie ForrestNicola J Stonehouse
Sep 10, 2014·Journal of Virological Methods·Fiona TullochMartin D Ryan
Oct 31, 2014·Journal of Virology·Yi-Ping LiJens Bukh
Mar 10, 2015·International Journal of Molecular Sciences·Huiding XieXiaoguang Xie
Citations
Mar 15, 2016·Scientific Reports·H StewartM Harris
Jul 28, 2016·Viruses·Siân CulleyJoe Grove
Jun 22, 2016·The Journal of General Virology·Eleni-Anna LoundrasNicola J Stonehouse
Dec 16, 2016·Cell Reports·Nicolai J Wewer AlbrechtsenJens J Holst
Dec 3, 2017·The Journal of Immunology : Official Journal of the American Association of Immunologists·Sarthak GuptaMariana J Kaplan
Nov 16, 2017·The Journal of General Virology·Niluka GoonawardaneMark Harris
Sep 13, 2018·ELife·Ying WangXiao-Ming Xu
Feb 28, 2019·PLoS Pathogens·Amina A NegashMichael Gale
Jan 21, 2018·PLoS Pathogens·Chunhong YinMark Harris
Sep 13, 2018·Scientific Reports·Christopher BartlettMark Harris
May 27, 2016·Journal of Virology·Jamel MankouriMark Harris
Sep 9, 2017·Neural Plasticity·Wen-Yuan LiZhen-Yu Wang
May 30, 2019·MBio·Sebastian WursterDimitrios P Kontoyiannis
Mar 23, 2018·International Journal of Molecular Sciences·Margarita-Maria PanouAndrew Macdonald
Nov 11, 2020·ELife·Joseph ShawStephen Griffin
Apr 2, 2020·Antiviral Research·Margarita-Maria PanouAndrew Macdonald
Mar 12, 2021·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Rongxin FuGuoliang Huang
Jul 16, 2021·PloS One·Jennifer J LabischKarl Pflanz