Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 3, Pages 1731-1741Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1912307117
Keywords
hepatitis E virus (HEV); infection; primary hepatocytes; humanized mice; transcriptomics
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Funding
- Boehringer Ingelheim Foundation
- Deutsche Forschungsgemeinschaft [398066876/GRK 2485/1]
- German Federal Ministry of Health [ZMVI1-2518FSB705]
- Research Foundation-Flanders [G0D2715N, G047417N]
- Research Foundation-Flanders (EOS project) [VirEOS30981113]
- European Union's Horizon 2020 research and innovation program [643476]
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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 culturederived particles (HEVcc) with viral titers between 10(5) and 10(6) 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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