Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway

Abstract

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. 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.

Selected Viruses Detected on and in our Food

“…HEV is the most important member of the family Hepeviridae and the genus Orthohepevirus. Interestingly, similar to HAV, hepatitis E virus particles are unenveloped when excreted in faeces but pseudo-enveloped in blood and cell culture [33]. HEV viruses infecting humans belong to the species Orthohepevirus A, whereas B, C and D infect birds, rodents and bats respectively [34].…”

Immunity against hepatitis E virus infection: Implications for therapy and vaccine development

“…However, a quasi‐enveloped virion is identified in HEV‐infected patients. HEV virions found in bile and feces are non‐enveloped, while those in the blood and culture supernatant are associated with lipids derived from the cellular membrane, thus resembling enveloped viruses . These lipid‐associated HEV virions are more resistant to anti‐HEV antibodies, suggesting HEV strategies to evade humoral immune responses.…”

Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

“…The disease is caused by infection with the hepatitis E virus (HEV), which is classified into the genus Orthohepevirus within the family Hepeviridae [4]. HEV is fecally excreted as a nonenveloped particle, but enveloped particles have been identified in serum and cell culture supernatants [5]. The genome is comprised of one single-stranded RNA molecule of positive polarity with a length of approximately 7 kb, containing three open reading frames (ORFs).…”

“…, lanes[5][6], indicating that no plasmid-derived sequences are added to the 3 -end of the generated virus genome.…”