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Review
. 2013 Feb;48(2):147-58.
doi: 10.1007/s00535-012-0682-0. Epub 2012 Oct 27.

Culture systems for hepatitis E virus

Hiroaki Okamoto  1
Affiliations
Review

Culture systems for hepatitis E virus

Hiroaki Okamoto. J Gastroenterol. 2013 Feb.

Abstract

The lack of an efficient cell culture system for hepatitis E virus (HEV) has greatly hampered detailed analyses of this virus. The first efficient cell culture systems for HEV that were developed were capable of secreting infectious HEV progenies in high titers into culture media, using PLC/PRF/5 cells derived from human hepatocellular carcinoma and A549 cells derived from human lung cancer as host cells. The success achieved with the original genotype 3 JE03-1760F strain has now been extended to various HEV strains in fecal and serum samples obtained from hepatitis E patients and to HEV strains in fecal and serum samples and liver tissues obtained from pigs and wild boar across species barriers. In addition, infectious HEV cDNA clones of the wild-type JE03-1760F strain and its variants have been engineered. Cell culture-generated HEV particles and those in circulating blood were found to be associated with lipids and open reading frame 3 (ORF3) protein, thereby likely contributing to the assembly and release of HEV from infected cells both in vivo and in vitro. The ORF3 protein interacts with the tumor susceptibility gene 101, a critical cellular protein required for the budding of enveloped viruses, through the Pro, Ser, Ala, and Pro (PSAP) motif in infected cells; ORF3 is co-localized with multivesicular bodies (MVBs) in the cytoplasm of infected cells, thus suggesting that HEV requires the MVB pathway for the egress of virus particles. This article reviews the development of efficient cell culture systems for a wide variety of infectious HEV strains obtained from humans, pigs, and wild boar, and also provides details of a new model for virion egress.

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Figures

Fig. 1
Fig. 1
Quantification of hepatitis E virus (HEV) RNA in culture supernatants of PLC/PRF/5 cells after inoculation of serum samples at a viral load on the order of 106 (upper panel) or 105 (lower panel) copies per well, containing HEV of the indicated genotype. The asterisks in parentheses indicate serum samples with anti-HEV antibodies. See reference [61] for further details
Fig. 2
Fig. 2
Schematic diagram of the “enveloped” and “non-enveloped” HEV particles in infected hosts. HEV particles excreted from the liver into the circulation are likely to be present as “enveloped” virus with cellular membranes and open reading frame 3 (ORF3) proteins on their surfaces, while the ORF3 protein and cellular membranes are dissociated from the virion after shedding in the bile duct, which contains detergent (deoxycholic acid) and then in the duodenum, which contains protease (trypsin), both secreted from the pancreas
Fig. 3
Fig. 3
Quantification of HEV RNA in the culture supernatants of A549 cells inoculated with homogenate samples of swine (sw) liver tissues (a) and wild boar (wb) liver tissues (b). Modified from reference [65]
Fig. 4
Fig. 4
Characterization of the ORF3 mutants of HEV. a Schematic illustration of the full-length cDNA clone of the HEV JE03-1760F strain (pJE03-1760F/wild-type [wt]) and its derivative mutants. The nucleotide (nt) sequence of nt 5121–5180 of the full-length cDNA clones of pJE03-1760F/wt and pJE03-1760F/∆ORF3 (∆ORF3, for simplicity) and the amino acid sequence of amino acid (aa) 91–102 of the ORF3 protein of the full-length cDNA clones of pJE03-1760F/wt and its Pro, Ser, Ala, and Pro (PSAP) mutant mutLSAL (pJE03-1760F/mutLSAL; mutLSAL, for simplicity) are aligned. Although not illustrated in the Fig., a Ser-to-Leu mutation at aa 87, which is identical to the majority of reported genotype 3 HEV strains, was also introduced in the mutLSAL. The stop codon of the ORF1 gene and the proposed initiation codons of the ORF2 and ORF3 genes are indicated by lines above the nucleotides. The dots represent nucleotides identical to those at the top (wild-type). The initiation site of subgenomic mRNA transcription [92] is depicted by a vertical line with an arrow facing right. The ∆ORF3 mutant was generated by mutating ATG (Met) to GCA (Ala) at the start codon of the ORF3 gene. The mutLSAL mutant was produced by converting CCC (Pro) to CTC (Leu) at the 95th codon and CCT (Pro) to CTT (Leu) at the 98th codon of the ORF3 gene. b HEV RNA in the culture supernatant (upper panel) and cell lysate (lower panel) of A549 cells inoculated with the culture supernatant of wild-type, ∆ORF3, or mutLSAL RNA-transfected PLC/PRF/5 cells was quantified by measuring the RNA titer by real-time reverse transcription-polymerase chain reaction (RT-PCR). c Sucrose density-gradient fractionation of HEV in culture supernatants from PLC/PRF/5 cells transfected with RNA transcripts of wild-type, ΔORF3, or mutLSAL. Modified from reference [71]
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