Isolation and Adaptation Characteristics of Hepatitis A Virus in Primary African Green Monkey Kidney Cells: Production of Antigen Useful for ELISAs Serology

Abstract

Four strains of hepatitis A virus (HAV) were isolated from four fecal samples of patients with type A hepatitis by using primary African green monkey kidney (PAGMK) cells or FRhK-4 cells. In all four samples viral antigen became detectable in PAGMK cells at the 3rd passage level after 9 weeks of incubation; detectable levels of antigen were reached earlier in FRhK-4 cells. An enzyme-linked immunosorbent assay (ELISA) was used to detect HAV antigen (HAV-Ag). Blocking experiments with negative and positive human sera and with paired marmoset sera established the identity of the virus. Infectious virus appeared to be both intracellular and extracellular. Although HAV-Ag could not be detected in culture medium by ELISA, the HAV infectivity titers of culture media were as high as those of cell-associated viruses (> 106 TCID50/0.2 ml). The passage procedure was simplified by using only virus isolated from cell-free medium as seed material, and the HAV strains were successfully propagated for 12 consecutive passages through PAGMK cells at 2-week intervals. The tissue-culture-produced HAV-Mg proved to be useful as a source of antigen in ELISA for detection of human anti-HAV IgG and IgM. The HAV strains adapted to PAGMK cells lost or decreased their ability to grow in FRhK-4 cells, while one strain adapted to FRhK-4 cells grew equally well in both cell systems.