The cultivation of human rotavirus, strain ‘Wa’, to high titer in cell culture and characterization of the viral structural polypeptides

Abstract

The structural proteins of the ‘Wa’ (serotype 2) strain of human rotavirus have not been described previously. Single-cycle virus growth in MA-104 cells using 5 μg/ml of trypsin in the growth medium was rapid with maximal viral yields (≈10 −6 PFU/ml) obtained 10–12 h post-infection. There was a continuous progression of cytopathic effect (CPE) from 6- to 50-h post-infection. Under conditions of multiple-cycle growth, a greater concentration of trypsin (40 μg/ml) in the growth medium was required to obtain rapid progression of CPE and production of a high titer (≈10 −7 PFU/ml) of infectious (double-shelled) virus. Single- and double-shelled virions were separated by isopycnic centrifugation in CsCl and analyzed by SDS-PAGE. Five proteins with molecular weights of 116,000, 92,000, 88,000, 84,000 and 41,000 were identified as components of the inner shell and four proteins with molecular weights of 60,000, 38,000, 32,000 and 27,000 were located in the outer shell.