Genome subunit reassortment among bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes

Abstract

A simple and rapid procedure for determining the genotypes of viruses has been applied to analysis of genome subunit reassortment in heterologous crosses of Batai virus, Bunyamwera virus, and Maguari virus, three members of the Bunyamwera serogroup of bunyaviruses. The procedure for determining genotype made use of specific molecular probes to identify the parental origin of the L and M RNA subunits. Complementary DNA copies of the three RNA segments of Bunyamwera virus were prepared by reverse transcription using synthetic oligonucleotide primers for first and second strand synthesis. The cDNA transcripts were inserted into a pBR322 vector and gene-specific probes prepared from nick-translated plasmid DNA. L and M gene-specific probes were identified which could unequivocally discriminate Bunyamwera virus genome subunits in a dot-hybridization test using cytoplasmic RNA extracts immobilised on nitrocellulose filters. None of the S gene-specific probes were sufficiently discriminatory for use in this test. Instead the parental origin of the S RNA subunit was inferred from the electrophoretic mobility of the virion N protein. It was observed that reassortment did not occur at random in heterologous crosses of ts mutants of the three viruses, and only the M RNA subunit appeared to segregate freely. However, unrestricted reassortment was observed when recombinant viruses with nonhomologous subunit combinations were used as the parental viruses. It was concluded, therefore, that restriction was mediated at the gene product level and that nonrandom reassortment was not due to incompatibility of genome subunits.