Single Strain Isolation Method for Cell Culture-Adapted
Hepatitis C Virus by End-Point Dilution and Infection
Nao Sugiyama1, Asako Murayama1, Ryosuke Suzuki1, Noriyuki Watanabe1, Masaaki Shiina2,
T. Jake Liang3, Takaji Wakita1, Takanobu Kato1*
1Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan, 2Department of Gastroenterology and Hepatology, Shin-Yurigaoka General Hospital,
Kawasaki, Kanagawa, Japan, 3Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland,
United States of America
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.
Citation: Sugiyama N, Murayama A, Suzuki R, Watanabe N, Shiina M, et al. (2014) Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-
Point Dilution and Infection. PLoS ONE 9(5): e98168. doi:10.1371/journal.pone.0098168
Editor: Birke Bartosch, Inserm, U1052, UMR 5286, France
Received February 5, 2014; Accepted April 29, 2014; Published May 21, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of
Health, Labour and Welfare of Japan, and from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Hepatitis C virus (HCV) is one of the most important pathogens
causing liver-related morbidity and mortality [1,2]. HCV is a
positive-stranded RNA virus belonging to the Flaviviridae family.
Its genome, about 9.6-kb long, consists of an open reading frame
(ORF) encoding a large polyprotein that is cleaved by cellular and
viral proteases into at least 10 structural and non-structural (NS)
proteins [3,4]. The structural proteins include core, E1 and E2,
which form virus particles. The NS proteins include p7, NS2, NS3,
NS4A, NS4B, NS5A and NS5B, which are associated with viral
For research into the HCV life cycle and development of
antivirals, in vitro models of this virus are indispensable. First, an
HCV subgenomic replicon system was used to examine HCV
replication in cell culture [5,6]. The HCV infectious step has been
assessed by an HCV pseudo-particle (HCVpp) system harboring
E1 and E2 glycoproteins [7,8]. This system enabled us to identify
several HCV receptors. Finally, to investigate other steps in the
HCV life cycle, an HCV cell culture system was developed with a
unique genotype 2a strain, JFH-1 . This strain is able to
replicate efficiently in culture cells, and its characteristics enabled
us to observe the whole life cycle of this virus in cell culture by
using cell-culture generated HCV (HCVcc) [10–12].
By modifying this system with CD81-lacking HuH-7-derived
cells, we established a novel system designated the single cycle
virus production assay, and this enabled us to estimate the
efficiency of each step of viral replication, infectious virus
production, secretion and infection [13–16]. However, virus
production levels of wild-type JFH-1 (JFH-1/wt) in these systems
are limited, and this shortage sometimes leads to difficulties in
experiments that require high viral concentrations. To overcome
these shortcomings, recent studies have identified several adaptive
or compensatory mutations that enhance viral production of JFH-
1 [17–24]. The contributions of these mutations to the viral life
cycle are not well defined. In this study, we isolated the cell
culture-adapted JFH-1 virus, which that can efficiently produce
progeny viruses by serial passaging of JFH-1 transfected Huh-7.5.1
cells, and evaluated the affected steps in the viral life cycle.
Materials and Methods
The HuH-7-derived cell lines Huh-7.5.1, provided by Francis
Chisari (Scripps Research Institute, La Jolla, CA), and Huh7-25,
which lacks CD81 expression, were cultured at 37uC in a 5% CO2
environment using Dulbecco’s Modified Eagle’s Medium contain-
PLOS ONE | www.plosone.org1 May 2014 | Volume 9 | Issue 5 | e98168
infectious virus . The ability of efficient virus production of
JFH-1/2G and JFH-1/6B may be attributable to this mutation.
In conclusion, we were able to successfully isolate 2 cell culture-
adapted variants that can produce 3 log-fold more progeny viruses
than JFH-1/wt, and identified the responsible mutations. The
strategy of single virus isolation by end-point dilution and infection
used in this study may be useful for identifying strains with unique
characteristics, such as robust virus production, from diverse
populations, and for identifying the responsible mutations for these
The authors wish to thank Dr. Francis V. Chisari (Scripps Research
Institute, La Jolla, CA) for providing the Huh-7.5.1 cell line.
Conceived and designed the experiments: TJL TW TK. Performed the
experiments: NS AM RS NW MS TK. Analyzed the data: NS AM TK.
Wrote the paper: NS AM MS TK.
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