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J Virol. 2019 Feb 5;93(4). pii: e01207-18. doi: 10.1128/JVI.01207-18. Print 2019 Feb 15.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes.

Capelli N1,2,3,4, Marion O2,3,4,5, Dubois M1,2,3,4, Allart S2,3,4,6, Bertrand-Michel J3,7, Lhomme S1,2,3,4, Abravanel F1,2,3,4, Izopet J1,2,3,4, Chapuy-Regaud S8,2,3,4.

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Department of Virology, CHU Purpan, Toulouse, France.
INSERM, Toulouse, France.
Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
CNRS, Toulouse, France.
Department of Nephrology and Organs Transplantation, CHU Rangueil, Toulouse, France.
Cell Imaging Facility, INSERM, Toulouse, France.
Metatoul-Lipidomic Platform, INSERM, Toulouse, France.
Department of Virology, CHU Purpan, Toulouse, France


Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.


TCID50 ; bile acid quantification; colocalization; hepatitis E virus; hepatocyte; lipid-associated particle; polarized cell; quasienveloped virus

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