Fig. 1.

Generation of membrane-associated quasi-enveloped and nonenveloped HEV virions from two different strains of genotype 3 HEV. In vitro-transcribed full-length genomic RNA from the genotype 3 HEV (strain Kernow-C1/P6) infectious clone was transfected into Huh-7 S10-3 liver cells. The culture supernatants of the transfected cells were collected and concentrated via ultracentrifugation as the virus stock of the quasi-enveloped eP6 virus. The eP6 virus was treated by detergents to produce the nonenveloped P6 virus stock. The 10% fecal suspension from a pig experimentally infected with HEV (strain US2) was purified via centrifugation and filtration to produce the nonenveloped US2 virus stock. The US2 virus was then used to infect HepG2 liver cells to generate the quasi-enveloped eUS2 virus stock. The virus stocks produced in this study were quantified by qRT-PCR to determine the genomic RNA copy numbers (A) and the infectious virus titer (TCID₅₀ per milliliter) in Huh-7 S10-3 cells (B). (C) Sucrose density gradient fractionation of the virus stocks produced in this study. (D) The virus stocks were analyzed via Western blot analyses using antibodies against HEV ORF2 protein and exosome markers (CD63 and Rab27a).