Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles-now considered "quasi-enveloped particles"-are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV
RNA and the viral capsid (ORF2) and ORF3
proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior
detergent and
protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a
lipid membrane.
After treatment with
detergent and
protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2
monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by
lipid membranes resembling those of exosomes, similar to enveloped viruses.IMPORTANCE
Hepatitis E, caused by HEV, is an important
infectious disease that is spreading worldwide. HEV
infection can cause acute or
fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after
organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a
lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV
infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.