Cargando…

Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection

The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concent...

Descripción completa

Detalles Bibliográficos
Autores principales: Sugiyama, Nao, Murayama, Asako, Suzuki, Ryosuke, Watanabe, Noriyuki, Shiina, Masaaki, Liang, T. Jake, Wakita, Takaji, Kato, Takanobu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029950/
https://www.ncbi.nlm.nih.gov/pubmed/24848954
http://dx.doi.org/10.1371/journal.pone.0098168
_version_ 1782317301586984960
author Sugiyama, Nao
Murayama, Asako
Suzuki, Ryosuke
Watanabe, Noriyuki
Shiina, Masaaki
Liang, T. Jake
Wakita, Takaji
Kato, Takanobu
author_facet Sugiyama, Nao
Murayama, Asako
Suzuki, Ryosuke
Watanabe, Noriyuki
Shiina, Masaaki
Liang, T. Jake
Wakita, Takaji
Kato, Takanobu
author_sort Sugiyama, Nao
collection PubMed
description The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.
format Online
Article
Text
id pubmed-4029950
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-40299502014-05-28 Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection Sugiyama, Nao Murayama, Asako Suzuki, Ryosuke Watanabe, Noriyuki Shiina, Masaaki Liang, T. Jake Wakita, Takaji Kato, Takanobu PLoS One Research Article The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics. Public Library of Science 2014-05-21 /pmc/articles/PMC4029950/ /pubmed/24848954 http://dx.doi.org/10.1371/journal.pone.0098168 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Sugiyama, Nao
Murayama, Asako
Suzuki, Ryosuke
Watanabe, Noriyuki
Shiina, Masaaki
Liang, T. Jake
Wakita, Takaji
Kato, Takanobu
Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title_full Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title_fullStr Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title_full_unstemmed Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title_short Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection
title_sort single strain isolation method for cell culture-adapted hepatitis c virus by end-point dilution and infection
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029950/
https://www.ncbi.nlm.nih.gov/pubmed/24848954
http://dx.doi.org/10.1371/journal.pone.0098168
work_keys_str_mv AT sugiyamanao singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT murayamaasako singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT suzukiryosuke singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT watanabenoriyuki singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT shiinamasaaki singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT liangtjake singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT wakitatakaji singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection
AT katotakanobu singlestrainisolationmethodforcellcultureadaptedhepatitiscvirusbyendpointdilutionandinfection