scholarly journals Robust hepatitis E virus infection and transcriptional response in human hepatocytes

2020 ◽  
Vol 117 (3) ◽  
pp. 1731-1741 ◽  
Author(s):  
Daniel Todt ◽  
Martina Friesland ◽  
Nora Moeller ◽  
Dimas Praditya ◽  
Volker Kinast ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
Hepatoma Cell ◽  
Hepatitis E ◽  
Human Hepatocytes ◽  
Cell Culture Model ◽  
Genotype 3 ◽  
Human Hepatoma Cell ◽  
Culture Model ◽  
Viral Titers

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral–host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

10.3791/61373 ◽  
2020 ◽  
Author(s):  
Toni Luise Meister ◽  
Mara Klöhn ◽  
Eike Steinmann ◽  
Daniel Todt
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
High Titer ◽  
Hepatitis E ◽  
Cell Culture Model ◽  
Culture Model ◽  
A Cell

scholarly journals Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

2021 ◽  
Vol 9 (11) ◽  
pp. 2302
Author(s):  
Katja Schilling-Loeffler ◽  
Oliver Viera-Segura ◽  
Victor Max Corman ◽  
Julia Schneider ◽  
Ashish K. Gadicherla ◽  
...  
Keyword(s):  
Cell Culture ◽  
Wild Boar ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Enriched Library ◽  
Genome Copy ◽  
Genotype 3 ◽  
Wild Boars ◽  
Copy Numbers ◽  
Cell Culture Isolation

Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

scholarly journals Nirtetralin inhibits growth of hepatitis E virus in HepaRG™ cells

2015 ◽  
Vol 10 (2) ◽  
pp. 377
Author(s):  
Wu-Jing Zhang ◽  
Ying-Bin Zhu ◽  
Guo-Dong Zhang
Keyword(s):  
Quantitative Analysis ◽  
Hepatitis E Virus ◽  
Hepatoma Cell ◽  
Hepatitis E ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Treatment Groups ◽  
Hepatoma Cell Lines ◽  
The Core ◽  
Cell Refinement

<p>Among numerous established in human hepatoma cell lines, none has been shown susceptible to hepatitis E virus (HEV) infection. Differentiation and infect ability are maintained but when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. On exposure to the nirtetralin, the virion particles were found to be decreased with an IC<sub>50</sub> of 2.277. Quantitative analysis of total and closed circular HEV RNA by real-time PCR performed on five independent experiments showed that only 1-5% of the HEV RNA internalized at day 1 post-infection entered the core of the cell refinement. The knockdown of 4E-BP1 led to a 1.7 ± 0.6-fold (mean ± SD, n = 5, p&lt;0.01) and 2.4 ± 0.9-fold (mean ± SD, n = 4, p&lt;0.05) (by the clone 56) growth of HEV RNA, respectively. Duncan's multiple range tests were applied to compare the differences between the treatment groups.</p><p> </p>

Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines

2014 ◽  
Vol 185 ◽  
pp. 92-102 ◽  
Author(s):  
Suljid Jirintai ◽  
Tanggis ◽  
Mulyanto ◽  
Joseph Benedictus Suparyatmo ◽  
Masaharu Takahashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hepatitis E Virus ◽  
Hepatoma Cell ◽  
Rattus Rattus ◽  
Hepatitis E ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Hepatoma Cell Lines ◽  
Wild Rats

scholarly journals Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells

2009 ◽  
Vol 90 (2) ◽  
pp. 457-462 ◽  
Author(s):  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Yu Hoshino ◽  
Hideyuki Takahashi ◽  
Koji Ichiyama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cdna Clone ◽  
Hepatitis E Virus ◽  
Culture Supernatant ◽  
Cultured Cells ◽  
A549 Cells ◽  
Hepatitis E ◽  
Infectious Cdna Clone ◽  
Post Inoculation ◽  
Genotype 3

A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed in this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 107 copies ml−1 on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 106 copies ml−1 at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.

scholarly journals Thermal Stability of Hepatitis E Virus as Estimated by a Cell Culture Method

2016 ◽  
Vol 82 (14) ◽  
pp. 4225-4231 ◽  
Author(s):  
Reimar Johne ◽  
Eva Trojnar ◽  
Matthias Filter ◽  
Jörg Hofmann
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
Culture Method ◽  
Hepatitis E ◽  
Pathogen Transmission ◽  
Heat Inactivation ◽  
Virus Inactivation ◽  
Genotype 3 ◽  
The Future ◽  
A Cell

ABSTRACTHepatitis E virus (HEV) is an increasingly recognized zoonotic pathogen. Transmission is suspected to occur from infected pigs or wild boars to humans through direct contact, environmental pathways, or contaminated food. However, the physical and chemical stability of HEV is largely unknown, because suitable cell culture methods for infectivity measurement are missing. Here, we developed a titration method using infection of the cell line A549/D3 with HEV genotype 3 strain 47832c and subsequent counting of focus-forming units by immunofluorescence, which allowed HEV infectivity measurements within a 4-log-dilution range. Long-term storage of HEV in cell culture medium at different temperatures indicated a phase of rapid virus inactivation, followed by a slower progression of virus inactivation. Infective HEV was detected up to 21 days at 37°C, up to 28 days at room temperature, and until the end of the experiment (56 days) with a 2.7-log decrease of infectious virus at 4°C. Heat treatment for 1 min resulted in moderate decreases of infectivity up to 60°C, 2- to 3.5-log decreases between 65°C and 75°C, and no remaining virus was detected at temperatures of ≥80°C. Heating for 70°C resulted in a 3.6-log decrease after 1.5 min and the absence of detectable virus (>3.9-log decrease) after 2 min. The data were used to calculate predictive heat inactivation models for HEV. The results may help estimate HEV stability in the environment or food. The established method may be used to study other aspects of HEV stability in the future.IMPORTANCEIn this study, a cell culture method was developed which allows the measurement of hepatitis E virus (HEV) infectivity. Using this system, the stability of HEV at different time-temperature combinations was assessed, and a predictive model was established. The obtained data may help estimate HEV stability in the environment or food, thus enabling an assessment of the relative risks of HEV infection through distinct routes and by distinct types of food in the future.

scholarly journals Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination

2012 ◽  
Vol 86 (10) ◽  
pp. 5697-5707 ◽  
Author(s):  
P. Shukla ◽  
H. T. Nguyen ◽  
K. Faulk ◽  
K. Mather ◽  
U. Torian ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis E Virus ◽  
Human Gene ◽  
Gene Segment ◽  
Hepatitis E ◽  
Genotype 3

scholarly journals Upregulated MicroRNA-29a by Hepatitis B Virus X Protein Enhances Hepatoma Cell Migration by Targeting PTEN in Cell Culture Model

PLoS ONE ◽  
2011 ◽  
Vol 6 (5) ◽  
pp. e19518 ◽  
Author(s):  
Guangyao Kong ◽  
Junping Zhang ◽  
Shuai Zhang ◽  
Changliang Shan ◽  
Lihong Ye ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Migration ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepatoma Cell ◽  
Cell Culture Model ◽  
X Protein ◽  
Culture Model ◽  
B Virus

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

2007 ◽  
Vol 88 (3) ◽  
pp. 903-911 ◽  
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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