HuH-7-Lunet BLR Cells Propagate Rat Hepatitis E Virus (HEV) in a Cell Culture System Optimized for HEV

Abstract

The family Hepeviridae comprises the species Orthohepevirus A-D (HEV-A to -D). HEV-C genotype 1 (HEV-C1, rat HEV) is able to infect humans. This study investigated whether an optimized HEV-A cell culture system is able to propagate the cell culture-derived rat HEV, and if de novo isolation of the virus from rat liver is possible. We tested the liver carcinoma cell lines PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR for their susceptibility to HEV-C1 strains. Cells were infected with the cell culture-derived HEV-C1 strain R63 and rat liver-derived strain R68. Cells were maintained in MEMM medium, which was refreshed every 3-4 days. The viral load of HEV-C1 was determined by RT-qPCR in the supernatant and expressed as genome copies per mL (c/mL). Rat HEV replication was most efficient in the newly introduced HuH-7-Lunet BLR cell line. Even if the rat HEV isolate had been pre-adapted to PLC/PRF/5 by multiple passages, replication in HuH-7-Lunet BLR was still at least equally effective. Only HuH-7-Lunet BLR cells were susceptible to the isolation of HEV-C1 from the liver homogenate. These results suggest HuH-7-Lunet BLR as the most permissive cell line for rat HEV. Our HEV-C1 cell culture system may be useful for basic research, the animal-free generation of large amounts of the virus as well as for the testing of antiviral compounds and drugs.

Keywords: Orthohepevirus C; cell culture; hepatitis E virus; rat HEV; zoonosis.

Figure 1

Replication dynamics of the rat HEV (HEV-C1) in different human liver carcinoma cell lines. (A) PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR were inoculated with the PLC/PRF/5 cell culture supernatant positive for HEV-C1 strain R63. Supernatants of all three cell lines were collected on day 126 post inoculation and used to (B) passage the virus in a cell line-specific manner, meaning that the HEV-C1 strain R63 positive PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR cell culture supernatants were inoculated onto PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR, respectively. (C) The HEV-C1 strain R63 positive cell cultures from the first inoculation were expanded from T12.5 to T75 flasks and a second time from T75 to T175 flasks. As soon as the initial viral load was reached again in the supernatant, the cells were detached and aliquots stored at −192 °C. (D) R63 passage 6 (derived from PLC/PRF/5) and (E) R68 positive liver homogenate were also inoculated onto PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR.