Development and Characterization of a Genotype 4 Hepatitis E Virus Cell Culture System Using a HE-JF5/15F Strain Recovered from a Fulminant Hepatitis Patient

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA–transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

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Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

Viruses ◽

10.3390/v13030406 ◽

2021 ◽

Vol 13 (3) ◽

pp. 406

Author(s):

Marie Pellerin ◽

Edouard Hirchaud ◽

Yannick Blanchard ◽

Nicole Pavio ◽

Virginie Doceul

Keyword(s):

Cell Culture ◽

Culture System ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Inhibitory Effect ◽

Cell Culture System ◽

Efficient Replication ◽

A Cell ◽

Vitro Model

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.

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Development and evaluation of an efficient cell-culture system for Hepatitis E virus

Journal of General Virology ◽

10.1099/vir.0.82535-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 903-911 ◽

Cited By ~ 176

Author(s):

Toshinori Tanaka ◽

Masaharu Takahashi ◽

Eiji Kusano ◽

Hiroaki Okamoto

Keyword(s):

Cell Culture ◽

Culture System ◽

Hepatitis E Virus ◽

Culture Medium ◽

Hepatitis E ◽

Post Inoculation ◽

Cell Culture System ◽

Genotype 3 ◽

Serum Samples ◽

Efficient Cell Culture System

Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0×107 copies ml−1, genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4×104 copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1×105 copies ml−1 on day 60. When 8.6×105 copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6×107 copies ml−1 on day 60. HEV incubated at temperatures higher than 70 °C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 °C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.

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Prolonged Fecal Shedding of Hepatitis E Virus (HEV) during Sporadic Acute Hepatitis E: Evaluation of Infectivity of HEV in Fecal Specimens in a Cell Culture System

Journal of Clinical Microbiology ◽

10.1128/jcm.01086-07 ◽

2007 ◽

Vol 45 (11) ◽

pp. 3671-3679 ◽

Cited By ~ 63

Author(s):

M. Takahashi ◽

T. Tanaka ◽

M. Azuma ◽

E. Kusano ◽

T. Aikawa ◽

...

Keyword(s):

Cell Culture ◽

Acute Hepatitis ◽

Culture System ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Cell Culture System ◽

Fecal Shedding ◽

Sporadic Acute Hepatitis ◽

A Cell ◽

Acute Hepatitis E

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Cell culture system for hepatitis E virus

Uirusu ◽

10.2222/jsv.60.93 ◽

2010 ◽

Vol 60 (1) ◽

pp. 93-104 ◽

Cited By ~ 3

Author(s):

Hiroaki OKAMOTO

Keyword(s):

Cell Culture ◽

Culture System ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Cell Culture System

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Isolation of Subtype 3c, 3e and 3f-Like Hepatitis E Virus Strains Stably Replicating to High Viral Loads in an Optimized Cell Culture System

Viruses ◽

10.3390/v11060483 ◽

2019 ◽

Vol 11 (6) ◽

pp. 483 ◽

Cited By ~ 7

Author(s):

Mathias Schemmerer ◽

Reimar Johne ◽

Monika Erl ◽

Wolfgang Jilg ◽

Jürgen J. Wenzel

Keyword(s):

Cell Culture ◽

Culture System ◽

Hepatitis E Virus ◽

Calf Serum ◽

Hepatitis E ◽

Post Inoculation ◽

The Novel ◽

Cell Culture System ◽

Wild Type ◽

Viral Loads

The hepatitis E virus (HEV) is transmitted via the faecal–oral route in developing countries (genotypes 1 and 2) or through contaminated food and blood products worldwide (genotypes 3 and 4). In Europe, HEV subtypes 3c, 3e and 3f are predominant. HEV is the leading cause of acute hepatitis globally and immunocompromised patients are particularly at risk. Because of a lack of cell culture systems efficiently propagating wild-type viruses, research on HEV is mostly based on cell culture-adapted isolates carrying uncommon insertions in the hypervariable region (HVR). While optimizing the cell culture system using the cell culture-adapted HEV strain 47832c, we isolated three wild-type strains derived from clinical specimens representing the predominant spectrum of HEV in Europe. The novel isolates 14-16753 (3c), 14-22707 (3e) and 15-22016 (3f-like) replicate to high viral loads of 108, 109 and 106.5 HEV RNA copies/mL at 14 days post-inoculation, respectively. In addition, they could be kept as persistently infected cell cultures with constant high viral loads (~109 copies/mL) for more than a year. In contrast to the latest isolates 47832c, LBPR-0379 and Kernow-C1, the new isolates do not carry genome insertions in the HVR. Optimization of HEV cell culture identified amphotericin B, distinct salts and fetal calf serum (FCS) as important medium supplements. Overconfluent cell layers increased infectivity and virus production. PLC/PRF/5, HuH-7-Lunet BLR, A549 and HepG2/C3A supported replication with different efficiencies. The novel strains and optimized cell culture system may be useful for studies on the HEV life cycle, inactivation, specific drug and vaccine development.

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