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

Kentaro Yamada

Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi-Ken 329-0498, Japan

Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp

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.

Present address: Division of Infectious Diseases, Department of Social and Environmental Medicine, Institute of Scientific Research, Oita University, Oita 879-5593, Japan.

The nucleotide sequences reported in this study have been assigned DDBJ/EMBL/GenBank accession numbers AB437316 (pJE03-1760F/wt) and AB437319 (pJE03-1760F/ORF1).

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