Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells

Abstract

A human fecal isolate of hepatitis A virus strain HAS-15 was adapted to rapid growth in FRhK-4 cells by more than 20 7-day passages. A cell culture-derived inoculum of strain HAS-15 was used at a multiplicity of infection of 80 radioimmunofocus-forming units per cell, and a one-step growth curve was determined. Both intracellular production and supernatant release of infectious virions were evaluated. Detection of virus release into the medium directly corresponded to intracellular production of infectious virions. A classical eclipse period was not observed during the growth curve determinations; however, detectable infectious virion production was absent for approximately 20 h after infection. This 20-h period was immediately followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10(9) radioimmunofocus-forming units per ml was achieved, and this level remained essentially constant for up to 14 days after infection. The infectious virus and viral antigen produced during the growth cycle were ascertained by a radioimmunofocus assay and by a radioimmunoassay, respectively. Cell culture supernatants were negative for viral antigen as determined by the radioimmunoassay, even though as many as 10(8) hepatitis A virus radioimmunofocus-forming units per ml were found. An adsorption study was also performed with strain HAS-15 by using FRhK-4 cells. More than 99.9% of the infectious virus was adsorbed at 25 degrees C in less than 20 min.