Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems

Gastroenterology. 2018 Feb;154(3):663-674.e7. doi: 10.1053/j.gastro.2017.10.041. Epub 2017 Dec 24.


Background & aims: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells.

Methods: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies.

Results: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A.

Conclusions: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

Keywords: Antiviral; HLCs; Personalized Medicine; Primary Isolates.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / pharmacology
  • Cell Differentiation
  • Cyclophilin A / genetics
  • Cyclophilin A / metabolism
  • Drug Resistance, Viral
  • Genotype
  • Hep G2 Cells
  • Hepatitis E virus / drug effects
  • Hepatitis E virus / genetics
  • Hepatitis E virus / growth & development*
  • Hepatitis E virus / immunology
  • Hepatocytes / immunology
  • Hepatocytes / metabolism
  • Hepatocytes / virology*
  • Host-Pathogen Interactions
  • Human Embryonic Stem Cells / immunology
  • Human Embryonic Stem Cells / metabolism
  • Human Embryonic Stem Cells / virology*
  • Humans
  • Immunity, Innate
  • Induced Pluripotent Stem Cells / immunology
  • Induced Pluripotent Stem Cells / metabolism
  • Induced Pluripotent Stem Cells / virology*
  • Kinetics
  • Phenotype
  • RNA, Viral / genetics
  • Sofosbuvir / pharmacology
  • Time Factors
  • Transfection
  • Virus Replication* / drug effects


  • Antiviral Agents
  • RNA, Viral
  • Cyclophilin A
  • Sofosbuvir