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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

Hepatitis E in industrialized countries has not been well studied. To define the possible risk factors for transmission of hepatitis E virus (HEV) and for the severe form of hepatitis E in Japan, we investigated the clinical and virological characteristics of hepatitis E in 32 patients who contracted the mild (n=23) or severe form (n=9) of domestically acquired hepatitis E between 1996 and 2004 in Hokkaido, where hepatitis E is most prevalent in Japan. Nine patients with the severe form of hepatitis E included two patients with fulminant hepatitis E and seven patients who were diagnosed with severe acute hepatitis in which hepatic encephalopathy did not appear during the course of the illness despite low plasma prothrombin activity (or=20 mg/dl). At least 25 patients (78%) had consumed uncooked or undercooked pig liver and/or intestine 1-2 months before the onset of hepatitis E. When compared with the seven patients with HEV genotype 3, the 25 patients with HEV genotype 4 had a higher peak alanine aminotransferase (ALT) level (P=0.0338) and a lower level of lowest prothrombin activity (P=0.0340). The severe form of hepatitis E was associated with the presence of an underlying disease (56% [5/9] vs. 17% [4/23], P=0.0454). The study suggests that zoonotic food-borne transmission of HEV plays an important role in the occurrence of hepatitis E in Hokkaido, Japan, and that the HEV genotype and the presence of an underlying disease influence the severity of hepatitis E.

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Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs and wild boars to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. However, no efficient cell culture system for swine and boar HEV strains has been established. We inoculated A549 cells with 12 swine and boar HEV strains of liver, feces, or serum origin at an HEV load of ≥2.0 × 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~10(8) copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging from 1.0 × 10(2) to 1.1 × 10(7) copies/ml upon inoculation at a lower load of approximately 10(5) copies per well. All progeny were passaged successfully onto A549 and PLC/PRF/5 cells. In sharp contrast, no progeny viruses were detectable in the culture supernatant upon inoculation with 13 swine and boar HEV strains at an HEV load of <1.8 × 10(4) copies per well. The present study also demonstrates that swine liver sold as food can be infectious, supporting the risk of zoonotic food-borne HEV infection.