title: Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles.
abstract: OBJECTIVE
  An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection.
  
  
  METHODS
  We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy.
  
  
  RESULTS
  Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies.
  
  
  CONCLUSIONS
  These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sanada
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hirata
authors.affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Naito
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kikkawa
authors.affiliation: PhoenixBio Co, Ltd, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: PhoenixBio Co, Ltd, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Yamasaki
authors.affiliation: PhoenixBio Co, Ltd, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Chise
authors.initials: C
authors.lastname: Tateno
authors.affiliation: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ochiya
authors.affiliation: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Kohara
keywords: BSA, bovine serum albumin; ESCRT, endosomal sorting complexes required for transport; EV, extracellular vesicle; Extracellular Vesicles; GEq, genome equivalent; HA, hemagglutinin; HBIG, hepatitis B immune globulin; HBV; HBV, hepatitis B virus; HBc, hepatitis B core; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; MVB, multivesicular body; PBS, phosphate-buffered saline; PXB-cells, primary human hepatocytes derived from chimeric mice with human liver; STED, stimulated emission depletion; Transmission Pathway; anti-HBs, antibody to hepatitis B surface antigen; mRNA, messenger RNA; nSMase, neutral sphingomyelinase; nts, nucleotides; qPCR, quantitative real-time polymerase chain reaction