Cell culture systems for the study of hepatitis E virus

Meister, Toni Luise; Bruening, Janina; Todt, Daniel; Steinmann, Eike

doi:10.1016/j.antiviral.2019.01.007

Cited by 63 publications

(63 citation statements)

References 190 publications

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“…This long absence of in vitro systems also severely limited the development of effective antivirals and vaccines targeting HEV. Many different cell culture systems have been tested in the past using various HEV strains, but mostly viral replication progresses very slowly and infection with low virion counts often results in nonproductive infection (10)(11)(12). Recent breakthroughs have been achieved by identifying compatible cell lines and specific HEV strains (11).…”

Section: Significancementioning

confidence: 99%

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Todt

Friesland

Moeller

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Significancementioning

confidence: 99%

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Todt

Friesland

Moeller

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The strain harbors a specific genome insertion resulting from duplications of parts of its genome, which might confer efficient cell culture growth [13]. Another HEV strain very frequently used for studies involving cell culture and site-directed mutagenesis is the genotype 3a strain Kernow-C1 [8]. This strain also harbors an insertion of similar location and size as compared to strain 47832c.…”

Section: Discussionmentioning

confidence: 99%

“…Research on HEV is hampered by the lack of broadly applicable and efficient cell culture systems and tools for site-directed mutagenesis. Only a few genotype 3 strains have been reported to efficiently grow in cell culture [8,9]. Among them, the genotype 3a strain Kernow-C1 and the genotype 3b strain JE03-1760F have been used in several studies [8].…”

Section: Introductionmentioning

confidence: 99%

“…Only a few genotype 3 strains have been reported to efficiently grow in cell culture [8,9]. Among them, the genotype 3a strain Kernow-C1 and the genotype 3b strain JE03-1760F have been used in several studies [8]. For both of these strains, reverse genetics systems based on in vitro-transcribed RNA from infectious cDNA clones have been developed, which enable targeted genome manipulations [10,11].…”

Section: Introductionmentioning

confidence: 99%

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Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

Scholz

Bächlein²,

Gadicherla

et al. 2020

Pathogens

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The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.

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“…The study of HEV replication has been hampered for a prolonged period due to the lack of an efficient cell culture system that supports the replication HEV gts 1 to 4 . Non‐human primates (NHPs) are susceptible to infections with HEV gts 1 to 4 and 8 and pigs can be used for the propagation of HEV gt3 and 4 .…”

Section: Introductionmentioning

confidence: 99%

Updates in Hepatitis E virus (HEV) field; lessons learned from human liver chimeric mice

Sayed

Meuleman

2019

Reviews in Medical Virology

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Summary Hepatitis E virus (HEV) is the most common cause of viral hepatitis globally, and it is an emerging pathogen in developed countries. In vivo studies of HEV have long been hindered due to the lack of an efficient small animal model. Recently, human liver chimeric mice were described as an elegant model to study chronic HEV infection. HEV infection was established in mice with humanized liver that were challenged with stool preparations containing HEV genotype (gt)1 and/or gt3. An increase in viral load and the level of HEV Ag in mouse samples were markers of active infection. Plasma‐derived HEV preparations were less infectious. The kinetics of HEV ORF2 Ag during HEV infection and its impact on HEV diagnosis were described in this model. In addition, the nature of HEV particles and HEV ORF2 Ag were characterized. Moreover, humanized mice were used to study the impact of HEV infection on the hepatic innate transcriptome and evaluation of anti‐HEV therapies. This review highlights recent advances in the HEV field gathered from well‐established experimental mouse models, with an emphasis on this model as a tool for elucidating the course of HEV infection, the study of the HEV life cycle, the interaction of the virus with the host, and the evaluation of new anti‐HEV therapies.

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Cell culture systems for the study of hepatitis E virus

Cited by 63 publications

References 190 publications

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

Updates in Hepatitis E virus (HEV) field; lessons learned from human liver chimeric mice

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