Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation

Abstract

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 ؋ 10 4 copies per well and 100% at >3.5 ؋ 10 4 copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.Hepatitis E, an acute viral hepatitis caused by infection with hepatitis E virus (HEV), is a globally distributed human disease. In developing countries of Asia, Africa, and Latin America, where sanitation conditions are not well maintained, HEV infection is transmitted via the fecal-oral route through viruscontaminated water or food, with substantial mortality in pregnant women (7, 33). In industrialized countries, autochthonous hepatitis E is far more common than previously recognized and has a predilection for older men, in whom it causes substantial morbidity and mortality (5,13,31,36,44). HEV is the sole member of the genus Hepevirus within the family Hepeviridae (6). It is a single-stranded, positive-sense, polyadenylated RNA molecule of approximately 7.2 kb in size, with short 5Ј-and 3Ј-untranslated regions (53). The genomic RNA contains three open reading frames (ORFs). ORF1 encodes nonstructural proteins involved in virus replication and virus protein processing. ORF2 and ORF3 overlap, and the ORF2 and ORF3

A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells

“…Genotypes 2 and 4 HEVs have not yet been examined for the function of the ORF3 protein on virion egress by using infectious cDNA clones. However, given their conservation, and since the association of HEV virions of genotypes 1, 3 or 4 in blood circulation with ORF3 protein and lipids on the surface has been noted (Takahashi et al, 2010), it is very likely that the function of the ORF3 protein related to HEV morphogenesis is common to all HEV strains, irrespective of genotype.…”

“…Our previous study using an ORF3-deficient cDNA clone of pJE03-1760F/ wt revealed that the ORF3 protein is essential for virion egress from infected cells (Yamada et al, 2009a). Furthermore, the ORF3-null mutant produced virus particles with the same buoyant density (1.26-1.27 g ml 21 in sucrose) as faecal HEV, which were clearly different from the cell culture-generated wild-type virions and those in the circulation of infected individuals, which banded at 1.15-1.16 g ml 21 and are intimately associated with lipids and the ORF3 protein (Yamada et al, 2009a;Takahashi et al, 2008bTakahashi et al, , 2010. These observations suggest that the acquisition of host-cell membrane on the surface of the virions is dependent on the expression of the ORF3 protein.…”

“…Recently, using faecal suspensions or serum samples with high HEV loads originally obtained from hepatitis patients who contracted imported or domestic infection of genotypes 1, 3 or 4 HEV as inocula, we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5) and a lung cancer cell line (A549), which yielded an HEV load of up to 10 8 copies ml 21 in the culture supernatant, and successfully propagated multiple generations of serial passages in culture supernatant (Tanaka et al, , 2009Lorenzo et al, 2008;Takahashi et al, 2007Takahashi et al, , 2010. In addition, we constructed a full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 HEV that can grow as efficiently as the faeces-derived virus in both PLC/ PRF/5 and A549 cells (Yamada et al, 2009b).…”

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

“…Takahashi et al (24) recently showed that virtually any serum with a high HEV titer can infect cultured cells. RNA viruses exist as quasispecies, and, given the tremendous difficulties in developing a cell culture system for HEV, it appears that a sample with a high titer has an increased probability of containing a variant with the correct constellation of mutations needed to permit infection of a cultured cell.…”

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

“…HEV is a non-enveloped small virus with a diameter of 27-32 nm, present in the bile and faeces of infected hosts, while HEV particles in the circulating blood and culture supernatant are found to be associated with lipids (Takahashi et al, 2008b(Takahashi et al, , 2010Yamada et al, 2009a). The HEV genome is a positive-sense, ssRNA composed of approximately 7200 nt, which is capped and polyadenylated (Kabrane-Lazizi et al, 1999;Tam et al, 1991).…”