A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious v...

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Autores principales:	George Koutsoudakis, Sofía Pérez-del-Pulgar, Patricia González, Gonzalo Crespo, Miquel Navasa, Xavier Forns
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cb99fe127d4544c3bcc0ffcebba08a8d
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spelling	oai:doaj.org-article:cb99fe127d4544c3bcc0ffcebba08a8d2021-11-18T08:03:01ZA Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.1932-620310.1371/journal.pone.0053254https://doaj.org/article/cb99fe127d4544c3bcc0ffcebba08a8d2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23300900/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.George KoutsoudakisSofía Pérez-del-PulgarPatricia GonzálezGonzalo CrespoMiquel NavasaXavier FornsPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e53254 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q George Koutsoudakis Sofía Pérez-del-Pulgar Patricia González Gonzalo Crespo Miquel Navasa Xavier Forns A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
description	Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.
format	article
author	George Koutsoudakis Sofía Pérez-del-Pulgar Patricia González Gonzalo Crespo Miquel Navasa Xavier Forns
author_facet	George Koutsoudakis Sofía Pérez-del-Pulgar Patricia González Gonzalo Crespo Miquel Navasa Xavier Forns
author_sort	George Koutsoudakis
title	A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
title_short	A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
title_full	A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
title_fullStr	A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
title_full_unstemmed	A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.
title_sort	gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis c virus.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/cb99fe127d4544c3bcc0ffcebba08a8d
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A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus.

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