Use of transcription probes for genotyping rotavirus reassortants

Radioactively labeled transcripts (probes) synthesized in vitro from rotavirus particles were employed to genotype rotavirus reassortants obtained from cells coinfected with a cultivatable bovine rotavirus (UK strain) and a noncultivatable human rotavirus (“Wa” or DS-1 strain). The technique consists of denaturing the genomic double-stranded RNA (ds RNA) from the reassortant and hybridizing it to labeled single-stranded RNA (ss [ 32P]RNA) prepared from either parent by in vitro transcription. Electrophoresis and autoradiography of the hybrids permit recognition of the parental origin of each of the genes of the reassortant. By using this approach, we have confirmed that gene 9 of the “Wa” virus codes for the specific antigen that reacts with neutralizing antibody. In addition, an analysis of a series of reassortants suggests that in the two human viruses studied, gene 4 may be responsible for restriction of growth in tissue culture.