Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures

Abstract

Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.