Development and evaluation of an efficient cell-culture system for Hepatitis E virus

1 Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan 2 Department of Nephrology, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan Correspondence Hiroaki Okamoto hokamoto{at}jichi.ac.jp Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0 x 10 7 copies ml –1 , genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4 x 10 4 copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1 x 10 5 copies ml –1 on day 60. When 8.6 x 10 5 copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6 x 10 7 copies ml –1 on day 60. HEV incubated at temperatures higher than 70 °C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 °C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.